Comprehensive Bioactive Natural Products, Volume 2: Efficacy, Safety & Chlinical Evaluation (Part I)

Comprehensive Bioactive Natural Products Volume 2

Efficacy, Safety & Clinical Evaluation I

V.K. GUPTA Indian Institute of Integrative Medicine (Council of Scientific & Industrial Research) Canal Road, Jammu (J&K State)- 180 001, India

2010

CID

Studium Press LLC, U.S.A.

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Comprehensive Bioactive Natural Products

VoL 2: Efficacy, Safety & Clinical Evaluation I © Copyright 2010

This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the editor and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. All rights are reserved under International and Pan-American Copyright Conventions. Apart from any fair dealing for the purpose of private study, research, criticism or review, as permitted under the Copyright Act, 1956, no part of this publication may be reproduced, stored in a retrieval system or transmitted, in any form or by any means-electronic, electrical, chemical, mechanical, optical, photocopying, recording or otherwise-without the prior permission of the copyright owner.

ISBN: 1-933699-52-3 SERIES ISBN: 1-933699-50-7

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STUDIUM PRESS, LLC P.O. Box-722200, Houston, Texas-77072, USA Tel. 713-541-9400; Fax:713-541-9401 E-mail: [email protected]

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Comprehensive Bioactive Natural Products: (Multi-Volume Set) Series Editor: V.K. Gupta E-mail: [email protected];[email protected] Volumes Published (2010) VoL 1: Potential & Challenges Ed. V.K Gupta Vol. 2: Efficacy, Safety & Clinical Evaluation I Ed. V.K Gupta Vol. 3: Efficacy, Safety & Clinical Evaluation II Ed. V.K Gupta Vol. 4: Antioxidants & Nutraceuticals Eds. V.K Gupta & Anil K Verma Vol. 5: Immune-modulation & Vaccine Adjuvants Ed. V.K Gupta Vol. 6: Extraction, Isolation & Characterization Eds. V.K Gupta, S.C. Taneja & B.D. Gupta Vol. 7: Structural Modifications & Drug Development Eds. V.K Gupta, S.C. Taneja & B.D. Gupta Vol. 8: Quality Control & Standardization Eds. V.K. Gupta, S.C. Taneja & B.D. Gupta

MEMBERS Dr. A. Panossian: Swedish Herbal Institute Research and Development, Kovlingevagen 21, Vallberga, SE-312 50, Sweden; E-mail: [email protected] Prof. Yu Zhao: Department ofTCM & Natural Drug Research, College of Pharmaceutical Sciences, Room 513, Zhejiang University, Zijingang Campus, 388 Yuhangtang Rd., Hangzhou 310058, China; Email: dryuzhao®Zju.edu.cn;[email protected] Prof. A. Evidente: Department of Organic Chemistry, Dipartimento di Scienze del Suolo, della Pianta, dell'Ambiente e delle Produzioni Animali, Universita di Napoli Federico II, Portici, Italy; E-mail: [email protected] Prof. Mirian Salvador: Instituto de Biotecnologia, Universidade de Caxias do SuI, Rua Francisco Getulio Vargas, 1130, CEP 95070-560, Caxias do SuI, Rio Grande do SuI, Brazil; E-mail: [email protected]

Dr. Gregory Beck: Department of Biology, University of Massachusetts Boston, 100 Morrissey Blvd., Boston, MA 02125-3393, 617-287-6619, 6684; E-mail: [email protected] Dr. Stephen M. Sagar: Departments of Oncology and Medicine, McMaster University, Hamilton, Ontario, Radiation Oncologist, Juravinski Regional Cancer Centre, Hamilton Health Sciences Centre. Associate Brain-Body Institute (St Josephs Health Care Centre and McMaster University); E-mail: [email protected] Dr. Anil K. Verma: Sr. Asstt. Professor of Zoology, Govt. (P.G.) College for Women, Gandhi Nagar, Jammu-180 001(J&K State), India; E-mail: [email protected] Prof. Ian Fraser Pryme: Dept. of Biomedicine, University of Bergen, Jonas Lies vei 91, N-5009 Bergen, Norway; E-mail: [email protected] Dr. Robert Frangez: Institute of Physiology, Pharmacology and Toxicology, Veterinary Faculty, University of Ljubljana, Slovenia; E-mail: [email protected] Dr. George Qian Li: Herbal Medicines Research and Education Centre Faculty of Pharmacy, The University of Sydney, NSW 2006, Australia, E-mail: [email protected] Prof. Yuji Nagashima: Department of Food Sciences and Technology, Tokyo University of Marine Sciences and Technology, Konan 4-5-7, Minato, Tokyo 108-8477, Japan; E-mail: [email protected] Prof. Pius Mpiana Tshimankinda: Departement de Chimie, Faculte des Sciences, Universite de Kinshasa, B.P. 190, Kinshasa XI, RD Congo; E-mail: [email protected] Prof. Supayang Piyawan Voravuthikunchai: Natural Products Research Center and Department of Microbiology, Faculty of Science, Prince of Songkla University, Hatyai, Songkla, Thailand - 90112; E-mail: [email protected]

SERIES ISBN: 1-933699-50-7

About the Series Nature abounds with a rich potential heritage ofbioactive natural products as that have been exploited for effective and beneficial use either as a therapeutic resource against a human disease or as a chemopreventive, antioxidant, nutraceutical or diet supplement. A natural product is a substance or a chemical compound produced by a living organism found in nature that usually has a pharmacological or biological activity. Some of these agents are derived from terrestrial plants, whereas, others are obtained from microorganisms, marine or freshwater organisms and even the higher animals. Past few decades have witnessed an unprecedented worldwide growth in the interest of bioactive natural products based medicines, both in developed and developing countries, and offer a resource-pool for lead identification in drug discovery owing to the reason that isolation of a novel molecule is easier than de novo synthesis, especially when the molecular structure of the compound is very complex. On account of such advantages, pharmaceutical companies are opting for bioactive natural products based drugs. More than 20 new drugs, launched world over between 2000 and 2005, originate from natural products. Scrutiny of medical indications by source of compounds has demonstrated that natural products and related drugs are used to treat 87% of all categorized human diseases (infectious and non-infectious). The development of new bioassay techniques, biotechnology methods, screening and high performance analytical methods have introduced new concepts and possibilities of rational drug design and drug discovery. Interest in bioactive natural products research is growing more strongly and can be attributed to several factors, including remarkable diversity of natural products, unmet therapeutic needs, the development of novel and sensitive techniques to detect, isolate, purify and structurally characterize these active constituents. This has opened opportunities for multidisciplinary research that joins the forces of natural product chemistry, molecular and cellular biology, synthetic and analytical chemistry, bio-chemistry, biotechnology, general biology, and pharmacology to exploit the vast diversity of bioactive natural products. Taking cognizance of these facts, we have initiated efforts in editing the present series "Comprehensive Bioactive Natural Products" and brought eight volumes (1 to 8) providing edited information from over 139 original research and review articles by eminent scientists and researchers

from India and abroad, representing 40 countries ofthe world on wide range oftopics in the area of natural products categorized under the themes viz., 1. Potential & Challenges 2. Efficacy, Safety & Clinical Evaluation I 3. Efficacy, Safety & Clinical Evaluation II 4. Antioxidants & Nutraceuticals 5. Immune-modulation & Vaccine Adjuvants 6. Extraction, Isolation & Characterization 7. Structural Modifications & Drug Development 8. Quality Control & Standardization

These volumes critically evaluate the present state-of-art, current status and future prospects in a well-illustrated manner. It is hoped that this series, which reflects the contributor's research results as well as world wide reviews will be widely read and used by all interested in these fields and shall open new vistas of research and academic pursuits for those engaged in the fields of bioactive natural products. I would like to take this opportunity to thank each of the authors who have contributed in the preparation of this book series.

Jammu, India V.K. Gupta Series Editor

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AMITY INSTITUTE FOR HERBAL AND BIOTECH PRODUCTS DEVELOPMENT - An Institution of Ritnand Balved Education Foundation - Thiruvananthapuram

Prof. (Dr.) P. PUSHPANGADAN, M.Sc. M.Phil. Ph.D., FBRS FES. FNRS, FNSE, FNESA, FNAASc, FNASc., (UN Equator Initiative Laureate) Director General & Senior Vice President, RBEF (Former Director, NBRI, Lucknow)

08-06-2009

Foreword to the Series Medicine among the ancient civilizations was a blend of Religion and Science. Natural products played an important role throughout the world in the prevention and treatment of diseases. These natural products originated from varied sources such as plants, animals, micro-organisms, marine organisms, terrestrial vertebrates and invertebrates. In man's quest for discovering new and potent remedies for diseases, he keenly observed his immediate neighbourhood and experimented not only with organic substances but also with in-organic materials such as metals, minerals, ores etc. Over the years, the accumulated medical wisdom was compiled into excellent treatises and these have later developed into the various classical systems of medicine associated with the ancient civilizations and cultures of the world. Apart from the classical systems of medicines, which by its nature and practice had universal applications, there existed another stream of medicine in the countryside, among the less privileged class of people, among the forest dwellers and among the nomadic tribes which were location specific, confined to small communities, tribes and families. These medical systems were mostly perpetuated by oral traditions. Nevertheless, the value of the oral traditions cannot be overlooked as these localized knowledge systems are veritable treasures of invaluable information on the properties and uses of hundreds of plant, animal and mineral products. Ethno medical information is now increasingly being utilized in the search for novel bioactive molecules from natural products. Several well known plants such as Licorice (Glycyrrhiza glabra) myrrh (Commiphora sps) and poppy capsule latex (Papaver somniferum) were mentioned in the clay tablets dating back to 2600 B.C, obtained from Mesopotamia. These plants and their derivatives are still used in various forms as herbal drugs as well as pure isolates in different systems of medicine. For instance,

codeine, papaverine, morphine etc isolated from P. somniferum are still used in modem medicine. Similarly hemisuccinate carbenoxolone sodium, a semi synthetic derivative of glycyrrhetic acid isolated from licorice is used for the treatment of gastric and duodenal ulcers. Plants, especially those with ethno medical history have been the major source ofbio active molecules. According to a study by Fabricant and Farnsworth (2001), out of 122 plant derived drugs, 80% had their origin from plants with ethno medical use. Since secondary metabolites from plants are products ofbiosynthetic reactions occurring in living systems, bioactive molecules from natural sources are excellent drug candidates as they are perceived to show more biological friendliness than synthetic drugs. The ever increasing popularity and acceptance of natural products have evinced an extra ordinary interest and scope in the subject in recent years. Compendium of Bioactive Natural Products- a series of eight books edited by Dr. V.K Gupta (Volumes 1,2,3 and 5) and Dr. V.K Gupta, Dr. S.C. Taneja, Dr. B.D. Gupta and Dr. A.K.Verma (Volumes 4,6,7 and 8), published by MIS. Studium press LLC, U.S.A are an excellent compilation, giving comprehensive and reliable data on the current development in the field ofbioactive natural products. This series contain 139 original research and review articles contributed by a galaxy of eminent scientists and academicians from 40 countries around the world. The editors have taken care to invite eminent scholars in the relevant areas to contribute each chapter in the series. The series deals with wide ranging topics related to natural products such as extraction, isolation and characterization, structural modifications and drug development, quality control and standardization and, immune modulation and vaccine adjuvants, antioxidants and nutraceuticals, efficacy, safety and clinical evaluation (Parts-1 and 2) and potentials and challenges. The editors of this series Dr. V.K Gupta, Dr. S.C. Taneja, Dr. B.D. Gupta and Dr. Anil K Verma are known to me for more than three decades and they are all stalwarts in natural products and related areas. It is gratifying to know that this series is dedicated to the memory of Col. Sir Ram Nath Chopra, the Founder Director of Regional Research Laboratory, now known as Indian Institute of Integrative Medicine (IIIM), Jammu.

Col. Sir R.N. Chopra (1882-1973) was a pioneer of systematic studies of indigenous drugs, promoter of Indian Systems of Medicine and Patron of Pharmacy in India. Most of the medicinal plants, he studied were in use, in Ayurveda for thousands of years. The major fields of Cot. Chopra's research were general pharmacology, chemotherapy, indigenous drugs, drug addiction and drug assays. At the Calcutta school of Tropical Medicine, he developed a pattern of research in studying the action of medicinal plants which influenced an entire generation of pharmacologists. Pharmacists and physiologists in India. In the words of Dr. G.V. Satyawati, Former Director General ofIndian Council of Medical Research, the credit of kindling the interest of Indian Chemists and Pharmacologists in Medicinal Plants should rightfully go to

Sir Ram Nath Chopra who, has been claimed as the Father of Indian Pharmacology' . I had the privilege and good fortune of meeting Col, Sir. Ram Nath Chopra along with Prof. Dr. E.K. Janaky Aromal at his residence in 1969 immediately after my joining Regional Research Laboratory as a Scientific Assistant. My meeting with Col. Sir R.N. Chopra had left a lasting impression of a great scientist in my memory. It is a fitting tribute to Col. Sir. R.N. Chopra that the editors have decided to dedicate these eight volumes to his memory. I congratulate the editors for bringing out this series which is timely, relevant and important to all those who are engaged in the study of natural products and development of therapeutically useful bioactive molecules. I am sanguine that these volumes will receive wider acceptance among the scientific community, especially among those engaged in Natural Product research.

(P. Pushpangadan)

About the Editor Dr. Vijay Kumar Gupta, Ph.D., FLS, London (born 1953-) Deputy Director & Head, Animal House, Indian Institute ofIntegrative Medicine (CSIR), Jammu, India. He did his M.Sc. (1975) and Ph.D. (1979) in Zoology both from University of Jammu, Jammu-India. His research capabilities are substantiated by his excellent work on histopathology, ecology and reproductive biology of fishes, turtles, birds and mammals, which has already got recognition in India and abroad. Dr. Gupta has to his credit more than 75 scientific publications and review articles which have appeared in internationally recognized Indian and foreign journals. Founder fellow, life member and office bearer of many national societies, academies and associations. He has successfully completed a number of research/consultancy projects funded by various governments, private and multinational agencies. His current areas of interest are histopathology, toxicology, pre-clinical safety pharmacology and reproductive efficacy studies oflaboratory animals. He is also Editor-in-chief of the books 1) Utilisation and Management of Medicinal Plants 2) Medicinal Plants: Phytochemistry, Pharmacology and Therapeutics 3) Perspectives in Animal Ecology and Reproduction (Vols.1-6). The Editor-in-chief of the American Biographical Institute, USA, has appointed him as Consulting Editor of The Contemporary Who's Who. Dr. Gupta also appointed as Nominee for the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA, Govt. oflndia). Recently the Linnaean Society ofLondon, U.K. has awarded fellowship to him in November 2009 in recognition of his contribution towards the cultivation of knowledge in Science of Natural History.

Preface The natural products have been of great help to mankind in resolving many diseases from earliest time. Primitive people in all ages have had some knowledge of natural medicines. The knowledge of the primitive man has been so modified with the advancement of civilization that know-a-days almost every type of medicine be that of Ayurvedic, Unani or even a number of allopathic medicines are obtained from natural sources. The medicinal value of drug plants is due to the presence of some chemical compounds in the plant tissue that produce a definite physiological action on human body. Early societies learned through trial and error that many plants contained substances with significant curative properties and over the years the natural products became used in more refined ways leading ultimately to the use of pure single component "active ingredient" as drugs. Nature has continuously provided mankind with a broad and structurally diverse array of pharmacologically active compounds that have proved to be indispensable for the cure of deadly diseases or as lead structures for novel pharmaceuticals. Their dominant role is evident in the approximately 60% of anticancer compounds and 75% of drugs for infectious diseases that are either natural products or natural product derivatives. At present 25% of the modern medicines are developed from plants that were first used traditionally, and many synthetic drugs have also been obtained from natural precursors. Almost, 70% modern medicines in India are derived from natural products. In the USA, the process of synthetic drug discovery and development takes an average of 12 years, and any new drug requires the investment of an average of US$ 230 million. It is seen that natural drugs take a comparatively much less time and expenses than synthetic drugs. Hence natural medicines would be cheaper, unless the market price are inflated by other considerations. Despite this success, during the past couple of decades, research into natural products has experienced a steady global decline. The introduction of high-throughput synthesis and combinatorial chemistry with their promise of a seemingly inexhaustible supply of compound libraries has greatly contributed to this declining interest in the screening of natural products by the pharmaceutical industry. Resistance of the parasites to existing drugs and their higher cost warrants the search of newer drug molecules. The origin of many effective drugs is found in the traditional medicine practices and in view of this several researchers have undertaken studies to clinically evaluate the medicinal plants for their preclinical safety and efficacy. The natural product market has been continually growing at an increased rate mainly as a nutraceuticals, dietary supplements, cosmoceuticals, chemopreventives and as herbal remedies. These therapies should emphasis on well-controlled and randomized clinical trials to prove

safety and efficacy and necessary guidelines should be followed in order to harmonies the use of bioactive natural products. At present, there is limited data on safety, efficacy and clinical trials to prescribe natural products in therapy. Realizing the need to document this knowledge gained through recent researchers, the present volume "Efficacy, Safety & Clinical Evaluation-I" of the book series entitled, "Comprehensive Bioactive Natural Products" presents 23 chapters discussing therapeutic potential of a large number of bioactive natural products of plant/animal origin investigated worldwide. Some of the interesting chapters included in this volume are: Perspectives in efficacy, safety and clinical evaluation of bioactive natural products; The modern application of traditional Chinese medicine: indigo naturalis as an alternative therapy in psoriasis vulgaris; Development of anti-angiogenesis therapies for treating cancer from Chinese medicinal herbs; Inflammation and mediators; Recommendation for reporting randomized controlled trials of herbal interventions: CONSORT for herbal medicine trials; Anxiolytic activity screening studies on extracts of a few medicinal plants; Larvicidal and antimicrobial activities of seeds ofAnnona cornifolia; Antidepressant activity of the extracts of three Hypericum species native to China; Cytolytic and toxic effects of ostreolysin, a protein from the oyster mushroom CPleurotus ostreatus); Antioxidant, antisecretory and gastroprotective activities from Leiothrix fiavescens; Evaluation of the immunity potency and security of inactivated oil-adjuvant vaccine against chlamydiosis in dairy cattle; Pharmacokinetics of active constituents of Rhodiola rosea SHR-5 extract; Metabolism and pharmacokinetic studies of coumarins; N aringin and its aglycone, research and managements; Biotransformation of drugs; Green tea: molecular targets in glucose uptake, inflammation, and insulin signaling pathways; Euphorbious plants as molluscicides and piscicides. The studies included are likely to lead further researches in this direction and it is hoped that this publication would attract world wide audience of researchers and the academicians of allied disciplines engaged in the search of new drug from natural resources.

Jammu, India

V.K.Gupta

ISBN: 1-933699-52-3

Table of Contents About the Series Foreword to the Series Preface Table of Contents of Volume 2 1.

Perspectives in Efficacy, Safety and Clinical Evaluation of Bioactive Natural Products

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1-29

YOGENDRANAYAK, VEERESHP. VEERAPUR, ANANTHA NAIK N AGAPPA AND M.K UNNIKRISHNAN (INDIA)

2.

The Modern Application of Traditional Chinese Medicine: Indigo N aturalis as an Alternative Therapy in Psoriasis vulgaris

31-43

YIN-Ku LIN AND JONG-HWEI Su PANG (TAIWAN)

3.

Development of Anti-Angiogenesis Therapies for Treating Cancer from Chinese Medicinal Herbs

45-65

STEPHEN M. SAGAR, RAIMOND K WONG AND DONALD R. YANCE JR. (CANADA, OREGON)

4.

An Anthology of the Studies on the Anti-Inflammatory, Antinociceptive and Febrifuge Effects of Medicinal Plants in Turkey

67-119

ESRA KUPEU AKKOL AND ERDEM YESILADA (TuRKEy)

5.

Comparative Study of the Antigenotoxic Effects of Some Selected Natural Plant Products Against Methylmethane Sulphonate Induced Genotoxic Damage in Cultured Mammalian Cells

121-131

YASIR HASAN SIDDIQUE AND MOHAMMAD AFZAL (INDIA)

6.

Inflammation and Mediators

A. KOUL, R. SHARMA, G.D. SINGH, A. KHAJuRIA AND V.K GUPTA (INDIA)

S. SINGH, P. KoUL,

133-178

7.

Recommendations for Reporting Randomized Controlled Trials of Herbal Interventions: CONSORT for Herbal Medicine Trials JOELJ. GAGNIER N.D., HEATHER BOON, PAULA ROCHON, DAVID MOHER, JOANNE BARNES AND CLAIRE BOMBARDIER FOR THE CONSORT GROUP (CANADA)

179-209

8.

Anxiolytic Activity Screening Studies on Extracts of a Few Medicinal Plants RICHA SHRI, MANJEET SINGH AND ANUPAM SHARMA (INDIA)

211-218

9.

Study of Eugeniajambolana and Momordica charantia in Respect to Safety and Antidiabetic Effect In vivo and In vitro YELE, SANTOSH AND VEERANJANEYULU, A. (INDIA)

219-230

10. Larvicidal and Antimicrobial Activities of Seeds of Annona cornifolia A. St. -Hil. (Annonaceae)

231-237

LUCIANA ALVES R.S. LIMA, MARIA AMELIA D. BOAVENTURA, JACQUELINE A. TAKAHASHI AND LUCIA P.S. PIMENTA (BRAZIL) 11. Antidepressant Activity of the Extracts of Three Hypericum species Native to China DEPO YANG, JIE BAr, ZHEN LI, GEORGE Q LI AND DONGMEI WANG (CHINA, AUSTRALIA)

239-249

12. Cytolytic and Toxic Effects of Ostreolysin, a Protein from the Oyster Mushroom (Pleurotus ostreatus)

251-264

KRIsTINA SEPCIC AND ROBERT FRANGE (REPUBLIC OF SLOVENIA) 13. Antioxidant, Antisecretory and Gastroprotective Activities from Leiothrix flavescens

265-279

THIAGO DE MELLO MORAEs, MARCELO APARECIDO SILVA, CLENILSON MARTINS RODRIGUES, LoURDES CAMPANER DOS SANTOS, MIRIAM SANNOMIYA, LUCIA REGINA MACHADO DA ROCHA, ALBA REGINA MONTEIRO SOUZA BRITO, TArS M. BAUAB, WAGNER VILEGAS AND CLELIAAKrKO HIRUMA-LIMA (BRAZIL) 14. Evaluation of the Immunity Potency and Security of Inactivated Oil-Adjuvant Vaccine against Chlamydiosis in Dairy Cattle Qru C., ZHOU J., CAO X. AND LIN G. (CHINA)

281-291

15. Annona crassiflora Wood Constituents: Antimalarial Efficacy, Larvicidal and Antimicrobial Activity

293-305

MARy ANE GONQALVES, VANESSA CARLA FURTADO MOSQUEIRA

AND

LUCIA PINHEIRO SANTOS PIMENTA (BRAZIL)

16. Pharmacokinetics of Active Constituents of Rhodiola rosea SHR-5 Extract A PANOSSIAN, A HOVHANNISYAN, H. ABRAHAMYAN, E. GABRIELYAN AND G. WIKMAN (ARMENIA, SWEDEN)

307-329

17. Metabolism and Pharmacokinetic Studies of Coumarins BEHERA D. AND AnDEPALLI V. (INDIA)

331-353

18. N aringin and its Aglycone, Research and Managements RICKYW.K WONG AND FANNYY.F. YOUNG (HONG KONG)

355-387

19. Biotransformation of Drugs M.G. TYAGI, S. PRABHAKARAN AND KR. DHANASEKHAR (INDIA)

389-398

20. Pharmacokinetics and Drug Interactions of Glycyrrhizin/Glycyrrhiza AK AGGARWAL AND D.K MAJuMDAR (INDIA)

399-416

21. Determination of Total Antioxidant Status and Rosmarinic Acid from Orthosiphon stamineus in Rat Plasma GABRIEL AKYIREM AKOWUAH AND ISMAIL ZHARI (MALAYSIA)

417-427

22. Green Tea: Molecular Targets in Glucose Uptake, Inflammation, and Insulin Signaling Pathways HEPING CAO, ANNE M. ROUSSEL AND RICHARD A ANDERSON (USA, FRANCE)

429-448

23. Euphorbious Plants as Molluscicides and Piscicides: A Review RAM P. YADAV AND AJAY SINGH (INDIA) Appendix

449-460

Table of Contents of Other Volumes of the SeriesVois. 1 & 3 to 8

461-474

Index

475-493

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1 Perspectives in Efficacy, Safety and Clinical Evaluation of Bioactive Natural Products YOGENDRA NAYAK1, VEERESH P. VEERAPUR2 , ANANTHA NAIK NAGAPPA3 AND M.K UNNIKRISHNAN 1

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ABSTRACT Natural products (NPs) include crude drugs, extracts, botanicals and pure compounds obtained from nature. NPs offer a rich source of diverse biologically active compounds with high molecular diversity, varying structural complexity and unparalleled novelty. A significant portion of the currently prescribed drugs owe their origin either directly or indirectly to NPs. The bioactivity of NPs not only builds the foundation for ethnic medicine systems of the world but also provides an impetus to modern drug discovery. Isolation of active component / molecule, structure elucidation, biological evaluation, dereplication, semisynthesis, biosynthesis, as well as optimization of downstream processing have undergone revolutionary changes in recent times, thereby enhancing the rate of drug discovery. The advent of high throughput screening and the availability of combinatorial compound libraries in natural products have been attracting major innovators towards natural products. Translational research has brought the drug discovery team closer to the clinical setting and has simultaneously increased the collective involvement of clinicians and pharmaceutical scientists. Complexity of natural products is fitly matched by the complexity 1. Department of Pharmacology, Manipal College of Pharmaceutical Sciences, Manipal University, Manipal- 576 104, India. 2. Department of Pharmaceutical Chemistry, SET's College of Pharmacy, S.R. Nagar, Dharwad - 580 002, India. 3. Department of Pharmaceutical Management, Manipal College of Pharmaceutical Sciences, Manipal University, Manipal- 576 104, India. * Corresponding author : E-mail: [email protected]

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of several pathological conditions, for instance diabetes, where multiple drug targets work in unison for producing a therapeutic outcome. Reverse pharmacology makes use of clinical evidences from traditional medicines, and then working backwards towards validation of claims in the laboratory. All the above themes are discussed in detail. Key words: Combinatorial compound library, drug discovery, high throughput screening, multiple drug targets, natural products, reverse pharmacology, translational research

IMPACT OF NATURAL PRODUCTS ON DRUG DISCOVERY: HISTORICAL PERSPECTIVES Natural products are rich sources of biologically active compounds with tremendous molecular diversity, possessing great promise in drug discovery and development (Butler, 2004; Newman et al., 2003a; Koehn et al., 2005; 2004b; Chin et al., 2006; Cragg et al., 2006; Blunt et al., 2007; Gordaliza, 2007; Butler et al., 2008). There is no dearth of illustrative examples to prove this paradigm. One of the oldest drugs, aspirin has had its origin from a plant (bark of willow), making it a historical land mark (Vane, 2000; Robert-II et al., 2001). Penicillin was first isolated from a microorganism Penicillum notatum (Wainwright, 1990). Morphine obtained from opium poppy is still an important drug in pain management. Bergmann's discovery and subsequent identification of spongothymidine and spongouridine in the early 1950s from the Caribbean sponge Tethya crypta led to the development of a nucleoside with biological activity (Bergmann, 1950). An explosive growth in synthetic chemistry further led to the discovery of a close analogue, cytosine arabinoside, a potent antileukemic agent (Beranek, 1986; Suckling, 1991). This compound was subsequently commercialized by Upjohn (now Pharmacia) as Ara-C. A closely related compound adenine arabinoside (Ara-A) was later synthesized and commercialized by Burroughs Wellcome (now Glaxo Smith Kline). Similarly, insulin was first isolated from the pancreatic extracts of animals such as cows, pigs and sheep, till recombinant DNA technology replaced the old technology. Eli Lilly (Indianapolis, USA) later demonstrated the commercial feasibility of recombinant DNA technology with the introduction of human insulin in 1982, ushering in a paradigm shift in drug discovery and development, leading to the start of the 'biotech era' (Pillai, 2001). The discovery of cyclosporine from microbial sources revolutionized immunosuppressive therapy for organ transplant patients (Kahan, 1999). Indeed, it has been estimated that at least 60% of the roughly 1184 new therapeutic agents introduced world-wide over the last twenty five years (1981-2006) have had their origins from natural products in one way or the other (Newman et al., 2007). The changing strategies and innovations in NP research have greatly influenced drug discovery programmes, in which

Efficacy, Safety and Clinical Evaluation of Bioactive Natural Products

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sample selection and collection, processing, isolation of active componenti molecule, structure elucidation, biological evaluation, dereplication, semisynthesis, biosynthesis, as well as optimization of downstream processing has become the norm. Such strategies have substantially enhanced the rate of discovery of novel NP with renewed interest in recent years (Gullo et al., 2005; Lawrence, 2005a, 2007b). With the advance of high throughput screening (HTS) and new instrumentation, the discovery of the new molecules with biological activity has hastened further. Newer guidelines and statutes implemented by various regulatory authorities have enhanced the time frame for the assessment of safety and efficacy of drugs in human populations. It can take up to fifteen years for the development of a molecule before it is commercialized into a marketed drug. Apart from the passage of time, a great amount of money is being spent in the process of developing a product into a clinically useful drug (Frank, 2003; Schmid et al., 2007). It is therefore important to examine the various events in drug discovery from natural sources and the associated regulatory rigors in assessment of quality, efficacy and safety of the drug. It is also essential to look into the translational aspects of NPs from preclinical to clinical studies (Dickson et al., 2004). The recent increase in the development-costs of new chemical entities (NCEs) has forced many innovators to search for active drug molecules from nature. The vast reservoir of diverse sources and the infinite variety in nature's molecular repertoire offers a fascinating arena for the drug discovery team.

NATURAL PRODUCTS FROM PLANT ORIGIN Medicines from plant sources have been used since ancient times for a variety of ailments. Herbal preparations have been used in the art of healing since time immemorial. Primitive man, through trial and error, gained knowledge of herbals and passed it on to their progeny. It appears that for thousands of years herbs were perhaps used for both their magical powers as well as medicinal values (Oumeish, 1998). Ancient civilization flourished in 3000 BC onward in Egypt, Middle East, India and China, with a parallel growth in the refinement of herbal medicine. The Egyptian Ebers Papyrus (1500 BC) is one of the earliest records providing information on herbs (Frey, 1986; EI-Gammal, 1994). In India, circa 1500 BC, Vedas included the Ayurveda system of medicine along with its vastly sophisticated informationbased on herbs, with about 350 herbals cited in this compilation (Patwardhan et al., 2004). Chinese traditional pharmacopoeia lists over 5700 traditional medicines which are mostly of herbal origin (Qiu, 2007). Chinese traditional medicine now enjoys credibility that is comparable to Western medicine in many parts of the world. Many universities in China teach and practice herbal medicine. Over the last 10 years Europe has witnessed a growth in the practice of Traditional Chinese Herbal Medicine.

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The development of organic chemistry has shown that the ability of herbal medicine to treat the body depended upon its chemical constituents. Chemists first began extracting and isolating chemicals from plants in the 18 th century. Encouraging results accelerated their enterprise and NP chemists have given to the world some of its most useful drugs such as morphine, digoxin, d-tubocurarine, ephedrine, quinine, vincristine, paclitaxel etc. A detailed list is presented in Table 1. Table 1. Prescription drugs derived from natural sources

Use Amino glycoside antibiotic

Analgesic

Drug name and the source Streptomycin (Bacteria; Streptomyces griseus) Gentamicin (Micromonospora) Kanamycin (Streptomyces kanamyceticus) Neomycin (Streptomyces fradiae) Netilimicin sulfate (Micromonospora) Morphine (Fruit exudate; Papaver somniferum)

Non-opioid analgesic

Epibatidine (Alkaloid that originally is found in the skin of a neotropical poisonous frog, Epipedobates tricolor); 200 times stronger than morphine; derivatives are under clinical investigation

Ansamycin antibiotic;

Rifamycin (Amycolatopsis rifamycinica); Anti-TB

Anti-Alzheimer's

Galantamine hydrobromide (An alkaloid obtained from Galanthus nivalis); Selective acetylcholinesterase inhibitor

Antibiotic

Mupirocin (Mixture of several pseudomonic acids obtained from Pseudomonas fluorescens); active against methicillin-resistant Staphylococcus aureus (MRSA) Chloramphenicol (bacterium; Streptomyces venezuelae) Tetracycline (polyketide antibiotic from Streptomyces) Daptomycin (Streptomyces roseosporus); Cyclic lipopeptide antibacterial agent for skin infections Teicoplanin (Actinoplanes teichomyceticus); Glycopeptide antiobiotic active against MRSA Vancomycin (Amycolatopsis orientalis); Glycopeptide antibiotic active against penicillin resistant Staphylococcus aureus Lincomycin (Streptomyces lincolnensis); Lincosamide antibiotic Erythromycin (actinomycete, Saccharopolyspora erythraea); Macrolide antibiotic Aztreonam (Chromobacterium violaceum); Monobactam antibiotic Cephalosporin (fungus; Cephalosporium acremonium); Beta lactum antibiotic Penicillin (Fungus; Penicillum notatum); Beta lactum antibiotic Colistin (polymyxin E) (Bacillus polymyxa var. colistinus); Polymyxin antibiotic Bacitracin (Bacillus subtilis var Tracy); Polypeptide antibiotic Fusidic acid (fungus: Fusidium coccineum); Urinary antibiotic

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Table 1. Contd. Use

Drug name and the source

Anticancer

Arglabin (from a species of wormwood Artemisia glabella endemic to the Karaganda region of Kazakstan) Bleomycin (Glycopeptide antibiotic from bacterium: Streptomyces vertic ill us) Etoposide (Herb: podophylum). Masoprocol (Nordihydroguaiaretic acid; NDGA; is a potent antioxidant compound found in the long-lived creosote bush: Larrea tridentata) Paclitaxel (plant; Taxus brevifolia) Solamargines (plant; Solanum marginatum) Vinblastin, vincristine (herb; Vinca rosea)

Anticancer antibiotic

Daunorubicin (Streptomyces peucetius) Pentostatin (Streptomyces antibioticus) Daunomycin (from bacterium: Streptomyces peucetius) Doxorubicin (Adriamycin; related to daunomycin from new strain of Streptomyces peucetius) Mithramycin (Plicamycin; Streptomyces plicatus) Mitomycin C (Streptomyces lavendulae) Sarkomycin (Streptomyces erythrochromogenes).

Anticoagulant

Heparin (sheep liver)

Antidiabetic

Insulin (pig and sheep) Voglibose (Aspergillus awamori)

Antifertility

Diosgenin (rhizome; Dioscorea deltoideae)

Antihypertensive

Reserpine (Rauwolfia serpentina)

Antimalaria

Quinine (Cinchona bark) Artemisinin (herb, Artemisia Annua)

Antiplatelet

Bivalirudin anew, genetically engineered form of hirudin, the substance in the saliva of the leech (Haementeria officinalis); used to reduce the risk of blood clotting in unstable angina who are undergoing a procedure to open blocked arteries in the heart.

Anti-spasmodic

Ephedrine (herb: Ephedra)

Cardiotonic

Digoxin (Fox glove; Digitalis species)

Hypolipidemic

Mevastatin (fungus; Penicillium citrinum)

Immunosuppressant for organ transplant

Sirolimus (Rapamycin; from bacterium Streptomyces hygroscopicus) Tacrolimus (bacterium Streptomyces tsukubaensis) Cyclosprin (fungus; Tolypocladium inflatum)

Local anaesthetic

Cocaine (plant; Erythroxylum coca)

Muscle relaxant

d-tubocurarine (plant; Chondrodendron tomentosum)

Mydriatic

Atropine (herb; Atropa belladonna)

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In spite of the great advances in synthetic and combinatorial chemistry, medicinal plants still make an important contribution in pharmaceutical innovation. According to the World Health Organization (WHO), about 65-80% of the world's population living in developing countries, still depends essentially on plants for primary health care because of poverty and lack of access to modern medicine (Pal et al., 2003). Some Western countries such as Germany, France, Italy and the United States have developed appropriate guidelines for registration of herbal medicines. Discovery of the blockbuster anticancer drug paclitaxel (Taxol) from the yew tree (Taxus brevifolia) once again helped to revive the interest towards herbal medicine (McChesney et al., 2007). Nowadays, several major pharmaceutical companies demonstrate renewed interest in investigating plants for discovering new sources for new lead structures and also for the development of standardized phytotherapeutic agents with improved efficacy, safety and quality (Gordaliza, 2007; Potterat et al., 2008).

NATURAL PRODUCTS FROM MARINE SOURCES Marine NPs have yielded a considerable number of new drug candidates which are still in preclinical or early clinical development (Haefner, 2003; Marris, 2006; Saleem et al., 2007). However a few are already in the market, such as cytarabine. Drugs from marine sources have a wide range of therapeutic benefits such as anticancer, antiviral, antiinfective, antimicrobial, analgesic, anti-inflammatory, anti-neurodegenerative in addition to many other therapeutic classes (Donia et al., 2003; Rawat et al., 2006; Bao et al., 2007; Diers et al., 2008; Uemura, 2006). Some of the NPs isolated from marine invertebrates have been shown to be, or are expected to be of microbial origin (Tan, 2007). Marine microorganisms, whose immense genetic and biochemical diversity is only beginning to be appreciated, appear to serve as a rich reservoir of NCEs. The prokaryotic marine cyanobacteria are a potential source of structurally bioactive secondary metabolites. A number of cytotoxic compounds such as hectochlorin, lyngbyabellins, apratoxins, and aurilides have been identified as potential lead compounds for the development of anticancer agents (Jimeno et al., 2004; Braiia et al., 2006).

NATURAL PRODUCTS FROM MICROORGANISM The seemingly endless potential of microorganisms dawned upon mankind in 1928 with Alexander Fleming's sensational discovery of an antibacterial filtrate "penicillin" from a commonplace mould Penicillium notatum (Ligon, 2004). Re-isolation and clinical studies pioneered by Chain, Florey, and co-workers in the early 1940s, and the subsequent scale-up towards commercially viable manufacture of synthetic penicillins revolutionized the foundation of antimicrobial drug discovery (Bentley, 2005). The

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phenomenal success of penicillin encouraged drug companies and research groups to pool their resources together for a large scale culture of a diverse array of microorganism from which several newer antibiotics emerged (Knight et al., 2003). Early years of this large scale hunt for antibiotics yielded a number of successful antibiotics, some of which are widely prescribed to this day. Prominent among these include pathbreaking molecules in the history of modern medicine, such as streptomycin, chloramphenicol, tetracycline, cephalosporin, erythromycin, and vancomycin. Each one of these compounds, either in its original form or in its synthetic derivative, can be found on the shelves of a pharmacy to this day. The impact of these drugs has even served to change the very attitude towards infective diseases from one of horror and despair to that of a routine ailment of trivial consequence. More lives have been saved in history by antibiotics derived from nature, than through any other class of prescribed drugs. In other words, NP derived drug molecules have contributed the most towards increasing the life expectancy of man. Antibiotics of natural origin also remain some of the most often prescribed drugs in the world. Some of the earliest compounds discovered in the early 1970s using rational approaches based on mechanism of action, include the betalactamase inhibitor clavulanic acid from Streptomyces clavuligerus (Brown et al., 1976) and the HMG-CoA reductase inhibitor mevastatin (earlier code-named as ML-236B) from Penicillium citrinum (Endo, 1981). A mixture of clavulanic acid and amoxicillin (the combination is called Augmentin) is being used today as a front line antibiotic in several critical infections, while mevastatin and lovastatin, have been the leads in the discovery of a series of drugs in the treatment of dyslipidemia. Today, these groups of statins are among the top revenue earners for some of the richest drug companies in the world (Manzoni et al., 2002). The arrival of statins has also widened the scope of therapeutic possibilities in compounds isolated from microbes. Many biotech industries are now utilizing microorganisms as the raw material for the production of not only antibiotics, but also various enzymes, antibodies, vaccines and many genetic engineered hormones.

EXPLOITING NEW APPROACHES FOR NATURAL PRODUCT BY PHARMACEUTICAL COMPANIES Just as the arrival of aspirin revolutionised the preparation of medicines from an individual enterprise of laboratory dispensing into a scale of industrial manufacture, the invention of penicillin and other antibiotics ushered in a new era of large-scale manufacture through biotechnology. Although 60% of the new drugs owe its origin to NPs, this enormous pool of resources is being steadily overlooked by several large pharmaceutical innovators for over a decade (Baker et al., 2007). This strategy seems

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inappropriate because several synthetic analogues of the NPs have often proved themselves to be more efficacious and less toxic. For instance, chloroquine is less toxic than quinine, and homatropine is shorter-acting and therefore produces less cycloplegia than atropine. Lignocaine, the more therapeutically useful product, was produced by modifying the cocaine molecule. Semisynthetic penicillins like methicillin expanded the range of cure. Similarly, second and third generation synthetic cephalosporins have extended the antibacterial spectrum of the prototype. The advent of high throughput screening (HTS, see below) and the availability of combinatorial compound libraries have been attributed as major reasons for this change in attitude (Boldi, 2004). NP-based drug discovery has been finding it difficult in adapting itself to modern techniques such as HTS and rapid hit-identification process. This disadvantage is being felt most acutely by large pharmaceutical companies with enormous funds at their disposal. The long time-gap between drug discovery and its marketing tends to challenge the wisdom in this tendency to move away from natural products. However, the scenario will become clear only in the long term. Interestingly, smaller biotech companies are emerging with renewed strategies to focus on exploiting new approaches for NP-based drug discovery. These approaches include the application of genomic tools, identification of novel sources of organisms, newer technologies (listed in the Table 2) and improved processes of sample preparation for screening. In addition, the recent focus on the synthesis of diversity-oriented combinatorial libraries based on NP-like compounds is an attempt to enhance the productivity of synthetic chemical libraries (Marcaurelle et al., 2008). One of the breakthroughs in drug discovery was the use of mechanism-based screening for bioassay-guided fractionation (Phillipson et al., 2002). Efforts continue to improve screening formats, reagent production, robotics, and data management. Mechanism-based screening Table 2. Technologiestl'echniques applied in drug discovery from natural products Name of the technology! technique Chromatography: High performance liquid chromatography (HPLC) High performance thin layer chromatography (HPTLC) Gas chromatography (GC) Liquid chromatography (LC)

Applications Isolation of components from the natural sources. In vitro and in vivo drug metabolism and pharmacokinetic (DMPK) assays, HTS new molecule identification, Biomarker identification (Korfmacher, 2005; Castro-Perez, 2007)

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Table 2. Contd. Name of the technology! technique

Applications

Supercritical fluid chromatography (SFC)

For the isolation of chiral compounds from natural sources (Zhang et al., 2005)

Mass Spectroscopy (MS): GC-MS, HPLC-MS, HPLCMS-MS, LC-MS, Fluorescence activated cell sorting (FACS)-MS, Liquid chromatography photodiode array mass spectrometry (LC-PDA-MS)

Molecular identification, and the structural elucidation of natural products usually combined with GC or HPLC or LC (Ma et al., 2006; Koehn, 2008)

FTICR-MS: Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometry

Biomarker discovery and analysis.Metabonomics, proteomics, natural product analysis and noncovalent complexes (including thermodynamics) (Feng et al., 2007; Zhang et al., 2005)

Nuclear magnetic resonance spectroscopy

Study on ligand binding to the protein, helps in the structure based drug design; Structural activity relationship (Robert, 2000; Coles et al., 2003; Pauli et al., 2005) Molecular isolation and identification usually combined with the HTS analysis (Koehn, 2008) For DMPK studies, HTS analysis of natural products (Corcoran et al., 2003) Studies of psychoactive agents in brain, Drug accumulation and metabolism in cancer, Quantitation and metabolism in the brain, liver and other organs (Paulus et al., 2007) To assess the biological function for unannotated proteins expressed by different genes with the protein structure solved (Reid et al., 2008; Powers et al., 2008)

(NMR)

Saturation Transfer Difference (STD)-NMR LC-NMR-MS, On-line solid-phase extraction (LC-SPE-NMR) Fluorine magnetic resonance (19F MR) Functional NMR (fNMR), Functional annotation screening technology by NMR (FAST-NMR) Positron emission tomography

Disease specific marker identification in nurological diseases (Wang et al., 2005; Beckmann et al., 2007; Hutchins et al., 2008)

High-throughput X-ray crystallography Wide-angle X-ray scattering

For protein structure studies, screening functional ligands, and ligand binding (Senior, 2005)

High-content highthroughput fluorescent microscopy

For the study of protein localization, dynamics and interactions with the natural products (Auer et al., 1998; Zemanova et al., 2003; Starkuviene et al., 2007)

has become the mainstay of HTS (Zheng et al., 2008). By utilizing new approaches, these companies are increasing the chemical diversity of their collections.

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SCREENING OF NATURAL PRODUCTS Traditionally, crude extracts have been used routinely for screening. Aqueous or alcoholic extracts are most frequently used for the initial round of biological and phytochemical screening. Recently developed bioactivity-guided fractionation processes can now separate crude fractions into mixtures containing only a few compounds. Column chromatography, highperformance liquid chromatography (HPLC), countercurrent chromatography (CCC), or centrifugal partition chromatography (CPC) are some of the techniques used for fractionation (Maugh, 1983). Fractionation will allow active compounds present in amounts too small to detect, whose activity is masked in a crude extract. However, by screening at higher concentrations, interfering compounds will also be more frequently identified. The paradox is that the total number of fractionated extracts screened is inversely proportional to the biodiversity. A decision must be made whether the loss of biological diversity is adequately compensated by fractionation. The answer to this question will help determine the optimal number of fractions that should be generated for each extract. The advent of sophisticated separation by analytical instruments attached to the HTS enables scientists to assemble large libraries of pure NPs, which can then be screened in a manner analogous to pure compound libraries (Liu et al., 2004). A recent review by McChesney et al. (2007) discusses latest methods and techniques involved in the natural product extraction, isolation and purification. The identification of the compounds that are responsible for the activity of an extract is called dereplication process (Konishi et al., 2007). This process prioritizes extracts for further study and can save considerable research time. The most generic procedure used today involves separation of an extract using reversed-phase HPLC and splitting of the eluent post column into a mass spectrometer (usually MSIMS to match fragmentation ions) and a fraction collector using micro-titer plates to test tubes, depending on the scale (Strege, 1998). The fractions are screened and the retention time, UV spectra, MS data, and activity of fractions are analyzed for common interfering compounds and known inhibitors using commercial and in-house databases (Schuffenhauer et al., 2005). Other methods for dereplication that have been used include separation by solid-phase extraction cartridges and CCC. Alternative methods for analysis include affinity capillary electrophoresis, LC-NMR, and online biochemical assay (Bobzin et al., 2000; Beger, 2006). An increase in the speed of bioassay-guided fractionation has been facilitated by a marked improvement in HPLC automation, MS, and column technology, as well as rapid turnaround of screening results provided by HTS (Mishra et al., 2008). The advent of new probe technology and higher magnetic fields has led to a significant shortening in acquisition time for

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NMR data, and the structure elucidation of NPs can be achieved routinely on amounts less than 1 mg (Liu et al., 2004; Xing et al., 2007). Automated structure-solving algorithms have made the job simpler but no technique can replace the structure elucidation skills of an experienced chemist (Ross et al., 2001). However, these programs add value to the tools that search for alternative structures that fit the same NMR data (Beger, 2006). IDGH THROUGH PUT SCREENING OF NATURAL PRODUCTS The discovery of truly novel NPs is a laborious and time consuming task when pursued through conventional programs such as extraction, fractionation, isolation and screening for biological activity. This has necessitated the adoption of high-throughput screening (HTS) by major players in the drug discovery team. HTS is defined as the automatic testing of potential drug candidates at a rate in excess of 10,000 compounds per week (Liu et al., 2004; Pereira et al., 2007; Koehn, 2008; Jenwitheesuk et al., 2008). HTS has gained widespread popularity over the last few years. The aim of high throughput drug discovery is to test large compound collections for potentially active compounds ('hits') in order to allow further development of compounds for pre-clinical testing ('leads'). In this approach, the molecular diversity and range of biological properties displayed by natural products is a challenge to combinatorial strategies for NPs synthesis and derivatisation (Posner, 2005). Many large companies study a hundred targets or more each year, and in order to validate these targets, lead compounds must be found. HTS is the process of assaying a large number of potential effectors of biological activity (lead) against the targets (a biological event) by miniaturized in vitro assays. Thus HTS technology has emerged over the last few years as an important tool for drug discovery and lead optimization in NP based drug discovery (Posner, 2005). HTS uses standard assay types known to most biological and biochemical scientists (e.g. ELISA, proliferation/cytotoxicity assays, reporter assays, and binding assays) (Inglese et al., 2007). The biochemical assay will be selected depending on the mechanism of action or target specificity of the compound or the mixture of compounds (commonly the fractionated extract). The primary assay is either cell free biochemical assays or cell based assays. Cell based assays allow the HTS to address a specific target (Castel et al., 2006). In order to process thousands of assays per day, HTS uses a combination of multiple well microplates and robotic processing. For a number of years, HTS assays have been run in the standard 96-well microplate (working volume of up to 250 ~L). The current goal of most companies is to move beyond this format to higher-density, lower-volume formats (e.g. 384 and 1536 well microplates). There are two primary advantages ofthese formats: increased throughput and lower volume, which

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translates into lower cost. At screening rates of 500,000 compounds/week, even a cost of $11 well becomes too big a burden on the company's weekly budget (Mishra et al., 2008). The reduction of cost, rather than increase in throughput, is the primary driving force within most HTS groups to move towards higher-density, lower-volume microplates. Fluorescence polarization (FP) detector technology is widely acceptable in the plate reader system (Burke et al., 2003; Rogers, 1997). For example screening assay for detection of apoptosis through activated caspase is assessed as liberation of free rhodamine following incubation of treated cells with rhodamine-labeled (quenched) substrate solution. Compounds showing a five fold or greater increase in fluorescence are considered as hits, being inducers of caspase activity. However, intrinsic fluorescence of natural products can interfere with such detection. Use of special reagents such as Europium-labeled reagents and time resolved fluorescence detection, avoids this problem (McMahon et al., 2000).

COMPOUND DEVELOPMENT: TOTAL SYNTHESIS, SEMISYNTHESIS OR COMBINATORIAL SYNTHESIS Perhaps the biggest obstacle to NP chemistry is the supply oflarge amounts of NPs. For instance, a steady supply of paclitaxel from nature would require cutting down a large number of whole yew trees followed by tedious extraction of the active molecule (McChesney et al., 2007). A sustainable source of the NP should be identified whenever total synthesis is not possible. However, a microbial source does not face such a problem on account of the brief life cycle of the organism and efficient culturing methods sustainable in lab conditions. This is precisely the reason why most companies prefer screening of microbial products. Rapid advances in microbial combinatorial biosynthesis have hastened the process in recent times (Dougherty et al., 2006; Adrio et al., 2006). There has also been progress with organisms usually considered to be problematic. For example, the steady supply of compounds from plants can be enhanced with minimal environmental damage through tissue culture and genomic methods (Wilkinson et al., 2005; Chaturvedi et al., 2007; GOmez-Galera et al., 2007). The study is now being extended to cover aquaculture of marine invertebrates and the identification, cloning, and expression of symbiotic microbial genes (Munro et al., 1999). The molecular complexity of NP derived NCEs can also impede the lead optimization process. A very important trend in recent years has been a significant effort devoted to the semisynthesis and synthesis of complex NPs (Ortholand et al., 2004; Shang et al., 2005; Paul et al., 2007; Bulger et al., 2008) (Listed Table 3). The success of this strategy is evident from history. Literature

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shows that a total of 322 NP-derived drugs have been launched from 1981 to 2006. More than 70% of these drugs (232 compounds) are manufactured by semisynthetic processes. Further, about 15% (47NCEs) have been produced by total synthesis (Newman et al., 2007; Butler, 2008). Paclitaxel, an anticancer drug manufactured by total synthesis, artemether, a semisynthetic drug from artemisinin, have emerged as synthetic breakthrough while vinblastine and vincristine (approved for cancer treatment in 1963 and 1965) templates are extremely difficult to synthesize economically. Table 3. Synthetic/semisynthetic analogues of natural products Name of drug

SourcelIndication

Acarbose

Hypoglycemic drug derivative of natural product from Aspergillus awamori

Alitretinoin

Anticancer; a first generation retinoid

Amrubicin

Derivative of doxorubicin from Streptomyces peucetius var caesius. Used in breast, lung, and gastric cancer

Anidulafungin

Antifungal agent; semi-synthetic lipopeptide from a fermentation product of Aspergillus nidulans.

Apomorphine hydrochloride

Synthetic analogue of morphine in Parkinson's disease

Arteether, artemether, artenusate

Semisynthetic derivative of artemesinin (from the plant Artemisia annual to treat malaria

Azithromycin

Macrolide antibiotic; Azane-substituent of erythromycin obtained from actinomycete Saccharopolyspora erythraea

Captopril, enalapril and cilazapril

Antihypertensive, developed from teprotide, isolated from Brazilian viper venom (Bothrops jararaca)

Carbepenams: Biapenem, Imipenam, Doripenem, Meropenam, Ertapenem Carumonam

Derived from a compound called thienamycin, produced by Streptomyces cattleya

Caspofungin acetate

Monobactam antibiotic, very resistant to ~-lactamase; derivative of aztreonam obtained from Chromobacterium violaceum Semisynthetic lipopeptide derived from pneumocandin BO, a fermentation product of Glarea lozoyensis; antifungal

Caspofungin acetate

Semisynthetic derivative of pneumocandin produced by fungi: Zalerion arboricola

Cefditoren pivoxil

An oral prodrug of cefditoren, a derivative of cephalosporin isolated from Cephalosporium species

Cladribine

Anticancer; related to nucleoside adenoside

Clari thromycin, Telithromycin, Dirithromycin

Macrolide antibiotic; Semi-synthetic erythromycin

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Table 3. Contd.

Name of drug Clindamycin Cytarabine ocfosfate

SourcelIndication Semisynthetic derivative of lincomycin, produced by the actinobacterium Streptomyces lincolnensis. Derivative of spongothymidine obtained from Caribbean sponge

Dicoumarol

Anticoagulant, derivative of plant coumarine isolated from fungal infected hay

Docetaxel

Anticancer; paclitaxel derivative

Ellipticine, Celiptium Epirubicin

Semisynthetic derivative from plant Ochrosia elliptica 4' -epimer of doxorubicin, an anticancer drug

Epirubicin HCI

Anticancer antibiotic related to doxorubicin

Etoposide phosphate

Anticancer; semisynthetic derivative from podophyllotoxin, found in the American Mayapple (Podophylum peltatuml

Everolimus

Orally active 40-0-(2-hydroxyethyl) derivative of rapamycin, originally produced from Streptomyces hygroscopicus.

Exemestane, Formestane

Anticancer; aromatase inhibitor structurally related to the natural substrate androstenedione

Exenatide

Antidiabetic: a synthetic analog of exenadin-4, which was originally isolated as a 39 amino acid peptide from the saliva of the Gila monster (Heloderma suspectuml

Fosfomycin trometamol

Antibacterial; analogue of fosfomycin obtained from Streptomyces fradiae.

Idarubicin hydrochloride (4demethoxydaunorubicinl

Anticancer antibiotic; derivative of daunourubicin

Irinotecan, topotecan

Camptothecin derivatives for cancer

Isepamicin

Aminoglycoside antibiotic; derivative of gentamicin B from Micromonospora, a genus of Gram-positive bacteria widely available in water and soil

Ivermectin

Anti-parasitic; derivative of avermectin obtained from microbe Streptomyces avermitilis

Micafungin sodium

Antifungal agent of the echinocandin type obtained from the culture broth of the fungus Coleophoma empetri ,and inhibits ~-(1, 3l-D-glucan synthase of fungi.

Micronomicin sulfate

Aminoglycoside antibiotic; 6'-N-methyl derivative of gentamicin C Antidiabetic; Acarbose related drug

Miglitol Miglustat

For type I Gaucher disease. An analogue of nojirimycin isolated from the broth filtrate of Streptomyces lavendulae

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Table 3. Contd. Name of drug

SourcelIndication

Miokamycin

Derivative of macrolide antibiotic miodecamycin Al ; 9,3'diacetylmiodecamycin; has wide antimicrobial spectrum

Mitobronitol (1, 6-dibromo1, 6-dideoxy-d-mannitoll

Alkylating anticancer; derivative of mannitol

Mycophenolate sodium

Mycophenolic acid, originally purified from Penicillium brevicompactum, used for prophylaxis in renal transplant

Nandrolone phenylpropionate

Anticancer; Analogue of naturally occurring nandrolone in human

Nitisinone

Derivative of leptospermone, an important new class of herbicides from the bottlebrush plant (Callistemon citrinus)

Orlistatin

Antiobesity; Semisynthetic product from lipstatin (a natural product)

Peplomycin

Semisynthetic analogue of bleomycin from bacterium Streptomyces verticillus

Pimecrolimus

A novel analog of ascomycin, isolated as a fermentation product of Streptomyces hygroscopicus var ascomyceticus, for allergic contact dermatitis and atopic dermatitis

Pirarubicin

Anticancer antibiotic similar to daunorubicin

Rifampicin

Semisynthetic compound derived from rifamycin which is obtained from Amycolatopsis rifamycinica

Rosuvastatin calcium

Hypolipidemic drug; derivative ofmevastatin isolated from Penicillium citrznum and P. brevicompactum

Telithromycin

Semi-synthetic derivative of the 14-membered macrolide, erythromycin-A, isolated from Saccharopolyspora erythraea

Testolactone

Anticancer; derivative of progesterone

Tigecycline

9-tert-butyl-glycylamido derivative of minocycline, which is a semi-synthetic product of chlortetracycline isolated from Streptomyces aureofaciens.

Tiotropium bromide

Synthetic derivative of atropine from Atropa belladonna (Solanaceae) to treat COPD

Triproamylin acetate

Antidiabetic; amylin analog

Valrubicin

Anticancer antibiotic similar to doxorubicin

Vapreotide acetate

Anticancer; Somatostatin analogue

Vindesine and vinorelbine

Anticancer; semisynthetic derivatives of vinblastine from herb Vinca rosea

Ziconotide

Non opioid analgesic; synthetic form of one of the orconotoxins, obtained from cone snail (Conus magus) venom

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Synthetic effort has been particularly critical for NP-derived anticancer drugs where total synthesis has been crucial in lead optimization and for obtaining enough material for clinical trials. Synthetic chemists are actively engaged in such ventures where there are 15 derivatives of paclitaxel, 15 of camptothecin, 4 of combretastatin, 4 of vinca alkaloids and 2 of podophyllotoxin in the clinical trial process (Saklani et al., 2008). With the advent of combinatorial chemistry, a large number of compounds can be produced from a single lead discovered from the NPs (Lee et al., 2001; Nielsen, 2002; Boldi, 2004; Ganesan, 2004; Tan, 2004). Combinatorial libraries based on natural product scaffolds, analogs and derivatives are being currently pursued. For example, combinatorial chemistry techniques applied to scaffolds oftubulin inhibitors, have yielded second-generation paclitaxel analogs as anticancer drug candidates (Ladame et al., 2008). Further, compounds active against vancomycin-resistant bacteria have emerged from combinatorial libraries of vancomycin- dimers (Griffin et al., 2003). The combinatorial libraries of anticancer molecules designed to target receptor tyrosine kinase, rest on the evidences obtained from a natural product genistein (Kissau, 2003). Thousands of camptothecin derivatives have been synthesized with the combinatorial approach to target topoisomerase I. Topotecan and irinotecan are derivatives obtained in this manner (Li et al., 2006). In spite of all the sophistication, combinatorial chemistry has failed to deliver the high numbers of drug leads as was predicted. This is mainly because of the complexity and diversity of the NP scaffold. Limited success has paved way for "diversity oriented combinatorial synthesis" which proposes to produce molecules with greater structural complexity including enhanced stereo-chemical variations (Rouhi, 2003; Liao, 2003; Tan, 2005; Marcaurelle et al., 2008), but these approaches may have limited prospects in future compared to the natural product based combinatorial library (Koppitz et al., 2006).

TRANSLATIONAL RESEARCH IN ASSESSING SAFETY AND EFFICACY OF BIOACTIVE NATURAL PRODUCTS: FROM BENCH TO BED SIDE In drug discovery and development programs, a vast majority of tested compounds do not progress from laboratories to patients' bedsides. Historical evidence indicates that only 14% of the tested products entering phase I trials eventually obtain clearance from the FDA and enter the market (DiMasi et al., 2003). In recent times, chances of success have reduced to just 8% (Graul, 2007). This high attrition rate is being attributed to the knowledge gaps between in vitro data, preclinical data and the final clinical outcome (Eisenstein et al., 2008). The basic knowledge about the investigational molecule or the natural product is not being translated

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appropriately to the clinical setting. The failures are not being analysed effectively together in a concerted manner by the drug discovery team and the clinical team. A two-way dialogue between scientists operating in preclinical drug discovery and clinical developers who understand the pathology and its alleviation, are required if novel leads and biologicals are to become useful drugs. Translational research, which focuses on such themes, has emerged as a new discipline in this context. The recent surge in translational research is expected to reduce the gap between the initial stages of drug discovery and subsequent clinical development (O'Connell et al., 2006). The American Physiological Society (APS) has defined translational research as 'the transfer of knowledge gained from basic research to new and improved methods of preventing, diagnosing or treating disease, as well as the transfer of clinical insights into hypotheses that can be tested and validated in the basic research laboratory (O'Connell et al., 2006). It is a kind of iterative learning experience from experimentation in basic and clinical fields. Thus in NP drug discovery, translational research interlinks preclinical and clinical research, where experiments progress steadily from lead generation towards proof of concept (early-stage clinical studies) (Sultana et al., 2007). Further, translational research assumes a broad perspective, embracing diverse areas such as clinical demands, public health policies and economic compulsions (Trusheim et al., 2007). The preclinical studies carried out in animals can predict efficacy (confidence in rationale) and safety (confidence in safety) of compounds or the natural product extracts in early development. The animal models and biomarkers (see below) can only give a preliminary idea of clinical correlation. Further, this may be poorly correlated to clinical outcomes in man. The pharmacokinetics and pharmacodynamics (PK-PD) of the compound in humans, which becomes clear only after the first dose in healthy volunteers, will help in determining the dose and route of administration. Research in normal human volunteers and patients enables a clearer understanding of the biology and pathophysiology, and can therefore trigger innovations in in vitro and animal experiments that better mimic the clinical condition. This strategy is therefore likely to enhance confidence in the appropriateness of a given target and help in dose selection for subsequent trials (Sartor, 2003). Biomarkers are quantitative measures of biological effects that provide informative links between mechanism of action and clinical effectiveness (Wehling, 2006). Assessment of biomarkers can reduce the uncertainty around key risks in the drug development cycle (pharmacology, PK-PD, toxicity, safety etc.). Further, biomarkers reflect biological effects induced by disease! test-compound and are the main tools to predict and describe its efficacy and safety. Specific markers like serum enzymes (ALT,

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AST, Catalase, Alkaline phosphatase etc) and more general markers of disease-severity like C-reactive protein in inflammatory diseases are well known biomarkers. Histological parameters, imaging of tumors and the morphology of atherosclerosis, liver fat (safety biomarker), positron emission tomography (brain disorders) scans etc. are some disease-specific biomarkers (Colburn, 2003). In preclinical studies, death could be a biomarker in models of myocardial infarction, cancer, acute toxicity etc., being correlatable measures of efficacy. These biomarkers are used to achieve predictive power and this can vary from almost useless (e.g. serial brain slices etc., which can never be a human biomarker) to surrogate (e.g. LDL cholesterol, one of the few surrogates accepted by FDA), with most cases falling somewhere in between. Translational research initially was said to be bidirectional but it has got multidirectional approaches (Sonntag, 2005). It has an important role in identifYing new indications for established therapies - a process termed by some authors as 'indications discovery'. New drug target hypotheses can be generated through preclinical experiments on novel target organs. It can also result from clinical observations such as side effects or additional pharmacological effects beyond those expected from efficacy in the main indication (O'Connell et al., 2006). Sildenafil, a selective Phosphodiesterase5 inhibitor, is an example of a drug initially assessed for angina pectoris but was subsequently licensed for a completely different pathology; male erectile dysfunction and pulmonary artery hypertension (Ghofrani et al., 2006).

NATURAL PRODUCTS FOR TYPE 2 DIABETES: A CASE STUDY Diabetes mellitus is a group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. The prevalence of diabetes for all age-groups worldwide was estimated to be 2.8% in 2000 and 4.4% in 2030. The total number of people with diabetes is projected to rise from 171 million in 2000 to 366 million in 2030 (Wild et al., 2004). Type 2 diabetes (T2D) is prevalent in 90% of the patients with diabetes. By 2025, more than three-quarters of all persons with diabetes will reside in developing countries. India and China are leading this surge in diabetes, and sub-Saharan Mrica is currently at a lower prevalence rate (Dagogo-Jack, 2006). Poorly managed diabetes leads to several complications (e.g. end-stage renal failure, blindness, amputation and heart disease) that many developing countries are ill equipped to tackle (Home, 2003). Diabetes is a heterogeneous metabolic disorder characterized by a decline in insulin action (insulin resistance), followed by pancreatic beta cell dysfunction (Chiasson et al., 2004). Insulin resistance will be associated with common conditions such as metabolic syndrome, dislipidaemia, obesity,

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endocrine disorders and hypertension. Abnormal obesity, increased lipid profiles, atherosclerosis and hypertension are symptoms of the metabolic syndrome (Wild et al., 2007). Prescription drugs available today reduce blood glucose level either by increasing the tissue uptake or by increasing the insulin secretion. There is no successful drug to treat the metabolic complexes associated with diabetes (Matfin, 2008). A wide range of natural products are prescribed to the diabetic patient as nutraceuticals or dietary supplements for the intervention of disease, treatment and/or prevention (for more details http://www.naturaldatabase.com). Compounds of chromium, selenium, vanadium, herbal preparations of garlic, ginseng, a-lipoic acid and various mixed-herbal products are among the dietary supplements that are currently being used by many diabetics (Table 4). A recent review by Hays et al. (2008) discusses the rationale behind prescribing these natural products. Despite compelling evidence supporting an important role for dietary chromium in the restoration of normal glucose tolerance among chromium- deficient individuals, the ability of chromium to improve insulin action remains controversial (Balk et al., 2007). While a number of animal models have generally demonstrated anti-diabetic properties for ginseng, relatively few clinical studies in humans have been reported. Taken together, research data suggest that ginseng is likely to show a beneficial effect on certain metabolic parameters in T2D (Xie et al., 2005). However, it is difficult to make standardized clinical recommendations on account of the large reported variability in metabolic responses to different ginseng species, different batches of the same species, and different parts of the ginseng plant. On the whole, standardization of natural products remains a major bottleneck in the search for clinical evidence.

Table 4. Commonly used conventional and natural products for diabetes Prescription drugs

Natural products

Hypoglycemic agents Chlorpropamid Glimepiride Glipizide Glyburide Nateglinide Repaglinide Tolazamide Tolbutamide

Banaba (Lagerstroemia speciosal Bitter melon (Momordica charantial Fenugreek (TrigoneUa foenum-graecuml Gymnema (Gymnema sylvestre)

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Table 4. Contd. Prescription drugs

Natural Products

Insulin sensitizers Metformin Pioglitazone Rosigli tazone

American ginseng (Panax quinquefolius) Banaba (Lagerstroemia speciosa) Cassia cinnamon (Cinnamomum aromaticum) Chromium Ginseng (Panax ginseng) Prickly pear cactus (Opuntia ficus-indica) Soya (Glycine max) Vanadium

Carbohydrate absorption inhibitors Acarbose Miglitol

Miscellaneous Exenatide Pramlintide Sitaglitpin

Blond psyllium (Plantago ovata) Fenugreek (Trigonella foenum-graecum) Glucomannan (Amorphophallus konjac) Guar gum (Cyamopsis tetragonoloba) Oat bran (Avena sativa) Prickly pear cactus (Opuntia ficus-indica) Soy (Glycine max) a-lipoic acid Selenium Stevia (Stevia rebaudiana)

Numerous cell culture and in vivo animal studies have demonstrated that treatment with the water soluble antioxidant a-lipoic acid is associated with improved skeletal muscle glucose transport activity, reduced wholebody insulin resistance, and reduced oxidative stress. Although relatively few clinical trials have been reported, the combination of compelling in vitro and in vivo animal data in the presence of limited clinical data indicate the need for further research in human subjects while only supporting a general recommendation for a-lipoic acid supplementation (Bilska et al., 2005). Epidemiological studies on various populations living on a diet consisting of a high proportion of fatty fish (a rich source of omega-3 fatty acids) demonstrate a lower incidence of T2D compared to matched population groups. Positive changes in lipid profile and blood pressure demonstrated by epidemiological and other studies suggest that omega-3 fatty acid supplementation may reduce cardiovascular disease risk in T2D patients, even if no direct improvement in insulin or glucose homeostasis has been conclusively demonstrated (Lombardo et al., 2006). Many Indian medicinal plants have shown marked efficacy in the treatment of diabetes (Saxena et al., 2004; Modak et al., 2007). A recent review by MukheIjee et al. (2006) has discussed the Indian medicinal plants

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with antidiabetic activity. However, detailed clinical investigations on the simultaneous use of prescription drugs along with natural products and dietary supplements are required for ensuring clinical safety and efficacy. A thorough preclinical and clinical investigation is necessary for establishing evidence. Diabetes presents a singularly complex condition where multiple targets operate in conjunction with the very complex physiology of nutrition and energy homeostasis. The number of druggable targets in diabetes is steadily increasing, with more and more successful drug classes entering the market. For instance, the DPP-IV (Dipeptidyl peptidase IV) inhibitor sitagliptin has recently been launched. While synthetic compounds address these targets individually, natural products probably tackle them in a collective and perhaps more holistic fashion. Restoring the equilibrium in the disturbed scenario of metabolic syndrome is probably linked to the ability to manage multiple targets taken together. While the effect on each individual target is sub-clinical, the collective pharmacodynamic picture is likely to playa role in restoring the complex metabolic equilibrium. For instance, we have demonstrated in our labs that extracts of Dodonaea I ~ t ~

f

SpeCific gene expression in

adlpocytes

.t- FA uptake '" Lipolysis

~

c==z:> ~ FFA

~oo~ t

II

- - _.......-

Insulin

'---V

sensitizer

.

factors

~ I

Expressionl action

.

"

~+

of insulin resistance factors

e.g. Reslstin! TNF

e ROS viz., ·OH, O2• •, Lpx Glucose Uptake

Fig 1. Schematic representation of the possible mechanisms by which a typical NP affords antidiabetic activity (PPAR y. Peroxisome Proliferator-Activated Receptor-Gamma; FA: Fatty Acids; FFA: Free Fatty Acids; TNF: Tumour Necrosis Factor; PTP-1B: Protein Tyrosine Phosphatase 1B; GLP-l: Glucagonlike peptide-1B; GIP: Gastric inhibitory polypeptide); DPP IV: Dipeptidyl peptidase IV; ROS: Reactive Oxygen Species; ·OH: Hydroxyl Radical; O 2 • -: Superoxide Radical; Lpx: Lipoxide Radicals)

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viscosa and Ficus racemosa stem bark show antidiabetic activity through multimodal mechanisms in animal models. The possible mechanisms suggested are increase in binding of PPAR-y (Peroxisome ProliferatorActivated Receptor-gamma), inhibition of PTP-1B (Protein tyrosine phosphatase 1B), DPP-IV (Dipeptidyl peptidase IV), IX-amylase, IX-glucosidase, tissue glucose uptake and reduction in the oxidative stress (Unpublished data, See Fig 1). The pharmacodynamic complexity is fitly matched by the diversity of targets addressing euglycemia in the body. While restoring or correcting these anomalies individually, one at a time, is the objective of conventional antidiabetic therapy, herbal products appear to tackle many of these targets in a concerted manner, providing a more sustainable method with perhaps a lower incidence of adverse events (Veerapur, 2007). REVERSE PHARMACOLOGY: AN EFFECTIVE STRATEGY The beaten track of drug discovery rests on the paradigm that the course of innovation begins in the lab and ends in the clinic. Reverse pharmacology is a complete turnaround in strategy, where clinical evidence works at the root of drug discovery (Patwardhan et at., 2004). It is essentially a clinic to lab strategy, working backwards, where existing claims by traditional systems of medicine, such as Ayurveda, give clues to possible clinical value of existing formulations in traditional medicine. In such situations, clinical safety and proof of concept are the premises upon which the program is built, thus saving millions of dollars that are customarily spent on ensuring clinical safety and meeting regulatory rigours. Consequently, efficacy becomes a matter of validation, with the help of scientific evidence gathered from laboratory research. It is therefore a confluence of traditional wisdom, modern medicine and science. If exploited intelligently, it becomes a viable mode of drug discovery where holistic systems and traditional wisdom are integrated with laboratory evidence and modern science. The major advantage of reverse pharmacology is that it helps in reducing three major bottlenecks that plague the drug discovery programmes viz., time, money and toxicity. These bottlenecks are major hurdles in drug discovery impeding not only the pace of its progress but also increasing the costs. Increase in regulatory rigour has become one of the major reasons for the ever diminishing entry of NCEs. In this context, reverse pharmacology appears to be a viable alternate strategy. Several industries have realized the need for reinventing the drug discovery strategies for innovating with value added products.

Ayurveda and such other traditional systems of alternate medicine have a rich knowledge base with a vast materia medica consisting of thousands of plants, minerals, animal products used singly or in combinations, with various processes for the formulation of medicines. Most

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of these formulations and therapeutic strategies have survived on the basis of evidence that have sustained centuries of clinical experience. However, lack of standardization and validation continues to undermine the pharmacopoeias of alternate medicine. It is in this context that modern medicine and science can act as a complementary support for establishing proof of concept and validation of claims.

CONCLUSIONS The last three decades have witnessed an unprecedented worldwide growth in the interest in NP- based medicines, both in developed and developing countries. Historical evidence shows that NPs offer a resource-pool for lead identification in drug discovery. NPs have a reasonable likelihood of biological activity, and isolation of a novel molecule is easier than de novo synthesis, especially when the molecular structure of the compound is very complex. NP-derived molecules are amenable to semisynthetic modifications and derivatisation. Such compounds are also significantly more complex and novel than most synthetic compounds, in addition to being chirally pure. Chiral specificity is very much in tune with the physiology of the body, giving the drug candidates a far greater level of specificity than nonchiral molecules. On account of such advantages, pharmaceutical companies are opting for NP based drug-leads. At present there is limited data on safety, efficacy, and clinical trials to prescribe NPs in therapy. However, the NP market has been continually growing at an increased rate during the last 30 years, mainly as nutraceutical, dietary supplements, cosmoceuticals and a few of them as chemopreventives. NP based therapy should emphasise on well-controlled and randomized clinical trials to prove safety and efficacy. International regulatory guidelines have to be followed in order to harmonise the use ofNPs. In some countries, the newer strategies and guidelines have been tried for the NPs (Briggs, 2002; Barnes, 2003; Vaidya et at., 2007; Calapai, 2008). Reverse pharmacology, which is driven by clinical evidence drawn from ancient wisdom, rather than preclinical data, also offers a viable time and cost saving strategy. Emphasis should also be given to the production and genetic improvement (whenever possible) of medicinal plants and other NPs. Recombinant DNA technology offers opportunities to synthesise the cryptic molecules of nature in a simple environmental-friendly way. Further, the emphasis must shift towards discovering leads from unexplored realms of biodiversity (Harvey, 2000; Harvey, 2007). Alternative sources for natural products as also the techniques to save the extinction of biodiversity should be given due importance for sustaining the program (Kennedy, 2008). Finally, but not any less important, is the need for a more detailed legislation on intellectual property rights related to the marketing of NPs. All these strategies are urgently needed to avoid both business conflicts and territorial conflicts on biodiversity.

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2 The Modern Application of Traditional Chinese Medicine: Indigo N aturalis as an Alternative Therapy in Psoriasis vulgaris YIN-Ku LIN 1 ,2 AND JONG-HWEI Su PANG2 ,*

ABSTRACT

Psoriasis is a distressing, recurrent disease that significantly impairs the quality of life and has no permanent cure. Traditional Chinese medicine is an alternative method of therapy that can be used in the psoriasis treatment. It has been reported in Chinese literature that indigo naturalis exhibited potential anti-psoriatic effects in systemic therapy. However, its pharmacology was unclear and its gastrointestinal side effects limited its use in clinical practice. This article reviews and summarizes our experience of psoriasis therapy using Chinese herb prescription and the investigation of the anti-psoriatic mechanism of indigo naturalis. Indigo naturalis formulated in ointment has efficacy in treating psoriasis and can avoid adverse systemic effects. The possible mechanism of indigo naturalis action in treating psoriasis includes inhibiting hyperproliferation of epidermal keratinocytes and lymphocytes infiltration, as well as restoring epidermal differentiation and tight junctions in psoriatic skin. We believe that indigo naturalis will become a promising natural product that exerts great therapeutic potential as an alternative therapy in psonas£s. Key words: Traditional Chinese medicine, psoriasis, indigo naturalis, indirubin, keratinocyte, differentiation 1. Department of Traditional Chinese Medicine, Center for Traditional Chinese Medicine, Chang Gung Memorial Hospital, Keelung, 204, Taiwan. 2. Graduate Institute of Clinical Medical Sciences, Chang Gung University, Tao-Yuan, 333, Taiwan. * Corresponding author: E-mail: [email protected]

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INTRODUCTION Psoriasis is a chronic inflammatory disease of the skin characterized by generalized erythematous papules or well-demarcated erythematous plaques topped by silvery scales which vary in severity and clinical manifestation. The most common form, plaque psoriasis, affects 2-3% of the Caucasian population (Lebwohl, 2003) but only 0.3% of the Mongoloid population (Yip, 1984). Many factors such as genetic features, immunological disorders, biochemical changes, environmental effects, trauma, stress, endocrine features, infection, drugs and alcohol play important roles in the pathogenesis of psoriasis (Ortonne, 1996). Conventional treatment for psoriasis has several disadvantages, including disease rebound or worsening after treatment is stopped (e.g. steroid) and risk for organ toxicities, such as nephrotoxicity and hepatotoxicity (e.g. cyclosporine, methotrexate). The fear of adverse events in long-term use may lead to poor compliance and results in following flares. Therefore, it is important to explore an alternatively new intervention which is relatively safe and can prolong the remission duration of psoriasis or prevent progression. The earliest description of psoriasis in ancient Chinese medical literature may be found in the 5th century AD. Traditionally, Chinese herbal preparation has been extensively used to treat psoriasis both topically and orally for centuries, and produced promising clinical results (Bedi & Shenefelt, 2002). Most of the key actions of Chinese herbs commonly used in treating psoriasis reflect anti-inflammatory properties, modulation of cytokine production or inhibition of angiogenesis (Tse, 2003). All these actions are potentially relevant in reducing the severity of psoriasis. Nowadays, traditional Chinese medicine (TCM) is still one of the most frequently chosen alternative therapies that can be used in the treatment of psoriasis in China and Taiwan. However, its lack of randomized blinded control trials and its underlying mechanisms of action have not been systemically investigated (Koo & Desai, 2003). The theoretical system and the terminology ofTCM are quite different from those of Western medicine. The doctors examine their patients with four traditional diagnostic methods (inspection, auscultation, interrogation, and pulse-taking), determine the principle of treatment according to the differentiation of symptoms and signs, and prescribe a formula according to the principle of treatment. In general, standard TCM practice emphasizes the importance of using many herbs that are combined in different formulations for each individual patient, sometimes mixing more than 10 different herbs. The mixture of herbs prescribed varies individually depending on the subtype of psoriasis (''blood-heat'' type, "blood deficiency dryness" type, and ''blood stasis" type), which is determined in traditional Chinese by many findings, including the lesions of psoriasis, the pulse, and the condition of the tongue (Tse, 2003). This is vastly different from Western medicine, which uses therapeutic approaches that are standardized and mono-therapy, as well as emphasizing

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"average" efficacy in large, double-blinded, placebo-controlled studies. Due to the overwhelming variability in how patients are treated, both between TCM practitioners and within the practice of a single TCM practitioner, it is difficult to conduct controlled trials to study traditional approach of individual polypharmacy. Indigo naturalis is one of the Chinese herbal remedies that have been reported to exhibit potential anti-psoriatic efficacy (Yuan et a!., 1982). However, the poor solubility and low absorption of indigo naturalis, and the adverse effects associated with irritation of the gastrointestinal tract and liver function in long-term systemic use have been occasionally reported (Verucchi et al., 2002). In order to avoid the adverse systemic effects, but retain the demonstrated efficacy of indigo naturalis as an anti-psoriatic medicine, we initiated an alternative approach in 2003 by applying the drug topically on skin lesions. Within the last five years, we have been using indigo naturalis formulated in ointment topically to treat thousands of patients with psoriasis. We also used indigo naturalis as a mono-therapy in treating psoriasis and conducted randomized, observer-blinded, vehicle controlled, intra-patient comparison trials to evaluate the clinical efficacy and safety in patients with psoriasis treated with indigo naturalis. This unique mono-therapy, rather than individual polypharmacy that TCM used to emphasize, hence provided a compelling evidence of indigo naturalis for treating psoriasis. This review aims to provide our clinical experience for the efficacy, safety of indigo naturalis in topical treatment of psoriasis, and our studies on the pharmacology of indigo naturalis and its active components.

INDIGO NATURALlS'S PROFILE Indigo naturalis (Qing Dai) is a dark blue powder originating from the branches and leaves of various indigo-producing plants, such as Strobilanthes fiaccidifolius, Polygonum tinctorium, Baphicacanthus cusia Bremek, and Isatis indigotica Fort. The extraction process is based on the natural fermentation method in Taiwan. In brief, the freshly cut plants are soaked in water until they decompose and the contents of indigo naturalis are ready to be extracted. An adequate amount of limestone powder is then added and oxygen is forced into the liquid usually by stirring the soaking solution thoroughly until it turns from blackish green to deep red, extracted products contained in the foaming surface of the mixture are collected and air-dried for later use. Indigo naturalis was used as textile dyes, paint pigment and medicine in ancient China. Indigo naturalis and indigo concoctions are recorded as being applied externally or taken internally before the first century AD in China and India. Until the mid-twentieth century, when antibiotics and steroids came into the world, indigo naturalis was one of the most important

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and efficacious treatments to heal ailments of various infections and inflammatory diseases in China and Taiwan. There are many people today who still have faith in the powers of indigo naturalis to cure various difficult illnesses, such as cancer. In TCM, indigo naturalis has been described as possessing antiinflammatory, anti-microbial, analgesic and sedative properties, i.e. "clearing away heat, detoxicating, removing blood-heat and ecchymoses, purging liver-fire and arresting convulsion". Indigo naturalis has been used as a remedy for high fever in infectious sickness, infantile convulsions, epilepsy, aphtha, mumps, pharyngitis, tonsillitis, gingivitis, periodontitis, and various dermatoses, including eczema, eruption, erysipelas, carbuncles, furuncles and insect or snake bites. Modern research, particularly in China, is revealing that indigo naturalis, with effects of antivirus, and anti-bacteria, has also been used as an important source of anti-cancer drugs in China (Han, 1994). A well-studied example is Danggui Longhui Wan, a mixture of 11 herbal medicines traditionally utilized against certain types of leukemia. Only one of these ingredients, indigo naturalis, was found to carry the antileukemic activity (Xiao et al., 2002). Although indigo naturalis is mostly constituted of indigo blue, a minor constituent, indirubin, was identified as the active component (Hoessel et al., 1999).

TOPICALLY INDIGO NATURALIS IN PATIENTS WITH PSORIASIS Clinical Efficacy Since 2003, we began to use Chinese herbs topically to treat patients with psoriasis. Initially, we used a combination of three herbs, indigo naturalis, Scutellaria baicalensis-Georgi, and Cortes phellodendri, which was recommended for psoriatic treatment in TCM. The topical ointment combined mixed herbs and vaseline, yellow wax as well as olive oil. We found that many patients with psoriasis, after multiple treatment failures with conventional anti-psoriasis medications, showed clinical improvement after topical treatment with indigo naturalis composite ointment. In addition, remission time was found to last longer than their previous therapies. This novel finding was reported in the journal Pediatric Dermatology (Lin et al., 2006). An 8-year-old boy with widespread psoriasis showed remarkable improvement with 8 weeks of topical treatment with this ointment. The psoriatic lesions on his body disappeared completely and the total body surface area involvement (BSA) decreased from almost 80% to 0%. Remission has lasted for over 2 years. In past 6 years observation, only mild disease recurred with no more than 1% ofBSA was noticed in this patient which was immediately improved after re-treatment with indigo naturalis composite ointment. Because this formula consists of three herbs, it is difficult to conduct controlled trials and investigations of pharmacological mechanism. We reviewed the published literature, and

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found indigo naturalis and its active ingredients, indirubin, have been orally used to treat psoriasis with considerable effectiveness (Koo & Arain, 1998). Therefore, we initiated the indigo naturalis mono-therapy for psoriasis and we reported that two adult patients with severe, recalcitrant psoriasis were successfully treated with topical indigo naturalis in the journal Clinical and Experimental Dermatology (Lin et al., 2006). Two patients were male aged 45 and 36. One had an 8-year history of psoriasis and was presented with 40% BSA and a Psoriasis Area and Severity Index (PAS!) score of 28.8. The other patient had suffered psoriasis for 22 years. He was presented with more than 80% BSA and a PASI score in excess of 42. The treatment by indigo naturalis ointment resulted in an approximately 95% reduction in PASI scores and BSA in these two patients within 3 months (Fig 1).

Fig 1. Clinical improvement after the treatment with indigo naturalis. A 40-year-old man with a 22-year history of plaques-type psoriasis . (A) Severe plaques are salmon pink in color, covered by silvery scales on right arm at the initial visit; (B) the same arm after 12 weeks of treatment with indigo naturalis ointment

In order to provide evidence-based data for this alternative therapy in psoriasis, we designed a randomized, vehicle-controlled clinical trial to evaluate the efficacy and safety of topically applied indigo naturalis in treating plaque-type psoriasis. An 8-week pilot clinical trial was performed during April-June 2004 in our hospital. Fourteen patients (10 men and 4 women) with chronic plaque psoriasis were enrolled. The mean patient age was 35.8 years (range, 21-54 years) and the median disease duration

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was 10.8 years (range, 2-30 years). The median pretreatment PASI was 11.7, with a median BSA of 13.6%. The patients were topically treated with either indigo naturalis ointment or vehicle ointment on contralateral skin lesions daily for 8 weeks. Efficacy was evaluated on the basis ofthe clinical scores, including induration, scaling, erythema and clearing percentage. Ten patients completed the trial and the results demonstrated a significant reduction in clinical scores achieved by topically applied indigo naturalis ointment. This interesting result has been published in the journal Dermatology (Lin et al. , 2007). To further confirm the efficacy of indigo naturalis for psoriasis treatment, another randomized, observer-blinded, vehicle controlled, intra-patient comparative trial was conducted. Fortytwo (32 men, 10 women) outpatients with chronic plaque psoriasis were enrolled. The mean patient age was 34.6 years (range, 18-58 years) and the median disease duration was 10 years (range, 2-41 years). The median pretreatment PASI was 14.7, with a median BSA of 18%. Target lesions were located on the upper extremities (17 participants), lower extremities (21 participants), and trunk (4 participants). The patients applied either indigo naturalis ointment or vehicle ointment topically to each of two bilaterally symmetrical psoriatic plaque lesions for 12 weeks. There was no significant difference in score of area, redness, thickness, and scaling between lesions which were assigned for treatment by indigo naturalis and vehicle ointments in baseline. The outcomes were assessed with the sum of erythema, scaling, and in duration scores and the clearing percentage of the target plaque lesion by two blinded professional observers. A significant reduction in the sum of scaling, erythema and in duration scores (p
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other currently used topical medicine (e.g. corticosteroid) to further evaluate the response duration and the disease recurrence. Histological Response to Indigo Naturalis Treatment in Psoriatic Lesions Based on our histological examination of skin biopsies, we found a marked decrease in epidermal thickness and elongated rete ridges for the lesions treated with indigo naturalis compared with the untreated lesions (Fig 2). We suggested that the anti-psoriatic effect of indigo naturalis might be mediated by controlling hyperproliferation and/or abnormal differentiation of epidermal keratinocytes, and/or exhibiting immunomodulatory effects in psoriatic lesion. We analyzed and compared the expression of different marker proteins with respect to proliferation (Ki-67, PCNA), differentiation (involucrin, filaggrin), and inflammation (CD3) in psoriatic lesions treated with indigo naturalis ointment and vehicle ointment. Results from immunohistochemical analysis showed a marked decrease in the number of cells with positively Ki-67 -stained nuclei in the indigo naturalis ointment-treated lesions compared to that in vehicle ointment-treated lesions (Lin et al., 2007). Similar findings were also demontrated for PCNA marker (Lin et al., 2007). The increased and normalized expression of filaggrin was observed in the stratum coreum and granular layer of the skin lesions treated with the indigo naturalis ointment (Lin et al., 2007). Involucrin also expressed normally in the granular and upper spinous layers after indigo naturalis therapy (Lin et al., 2009). In contrast to the original psoriatic lesions, the number of positively CD-3-stained T lymphocytes was demonstrated to be significantly reduced in the epidermis and papillary dermis of indigo naturalis ointment-treated lesions (Lin et al., 2007). In addition, based on our histological examination of skin biopsies, we found the presence of wide intercellular clefts only in the epidermis of lesions before treatment and control lesions treated with the vehicle ointment, but not in lesions treated with indigo naturalis ointment. In the treated psoriatic lesions that showed good clinical improvement, the keratinocytes in the granular layers were found to adhere to each other tightly. This finding implied that the impaired epidermal tight junction of psoriatic lesions may recover as a result of indigo naturalis treatment. We analyzed the expression of different marker proteins with respect to tight junction function (such as daudin-I) in psoriatic lesions, and the results showed a significant increase and recovery after treatment with topical indigo naturalis ointment compared with vehicle ointment (manuscript submitted).

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Fig 2. Effect of topically applied indigo naturalis ointment on the histological change of psoriatic lesion. Skin biopsies taken at baseline and after 8 weeks of treatment were processed for histological examination by HE stain method. (A) Untreated psoriatic lesion. (B) Indigo naturalis-treated lesion. Original magnification: xlOO

In short, our pathological findings suggested that indigo naturalis has great potential values in the treatment of psoriasis by inhibiting hyperproliferation of epidermal keratinocytes and lymphocytes infiltration, as well as restoring epidermal differentiation and tight junctions in psoriatic skin. Whether these findings are simply a result of skin recovered from psoriasis after indigo naturalis treatment or indigo naturalis might directly modulate these changes in skin is waited to be clarified by investigating the pharmacology of indigo naturalis in much more details.

Adverse Events Indigo naturalis, as a crude extract, appears to be dark blue in color and has a slightly unpleasant odor. The indigo naturalis ointment might slightly

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stain the skin and clothing which can be cleaned thoroughly by common detergents. Repeated application has no significant effect on the skin color and does not change the skin appearance. However, either the color or smell of indigo naturalis may affect the compliance offew patients, therefore benefit comes along with longer treatment can not possibly attain for them. Among 10 patients who completed an 8-week pilot clinical trial, 2 patients reported a short period (3-5 days) of itching at the beginning of treatment. In the following treatment period, no further adverse events such as scaling, erythema, tenderness, vesicles or other serious problems were found (Lin et al., 2007). Another 12 weeks clinical trial, 4 of 34 patients who completed the treatment reported itching after applying the indigo naturalis ointment to their psoriatic plaque, but the itching was only present for a couple of days at the start of treatment, and no tenderness, vesicles or other serious problems were found in the subsequent treatment period (Lin et al., 2008). In these two study groups, there were no abnormalities seen in the liver function tests, renal function tests, and complete blood counts after trial. Similar results were obtained in our long-term follow-up of these patients for the past 5 years. In our past 5-years experience in clinic, there were no serious events such as hepatic toxicity, renal damage and gastrointestinal problems found in these studies. Hypersensitivity, however, may be the major adverse effect as we have noticed in clinic (less than 15%). Some patients developed erythrodema and itching after using topical indigo naturalis treatment for psoriasis and most of them could recover by discontinuing the indigo naturalis application. Combining the treatment with topical steroids intermittently the development of allergy contact dermatitis may also be avoided or diminished.

PHARMACOLOGICAL PROPERTIES OF INDIGO NATURALIS Although the effectiveness of indigo naturalis in the treatment of psoriasis is well established, the mechanism of its action is poorly understood. We have utilized cultured normal human epidermal keratinocytes (NHEKs) as a psoriasis-relevant experimental model to investigate the potential effects of indigo naturalis on the gene expression of keratinocytes. In our laboratory data, we found that indigo naturalis could suppress proliferation and promote differentiation of epidermal keratinocytes which were correlated with the down-rgulation of PCNA gene expression and the upregulation of involucrin gene expression of NHEKs (Lin et al., 2009). We have also made another novel finding that indigo naturalis can improve skin barrier protection, at least in part, by up-regulating claudin-1 gene expression and therefore the tight junction function (manuscript submitted). The main components of indigo naturalis used in our clinic are indigo blue, indirubin and tryptanthrin as identified in chromatographic fingerprints. Among them, indigo blue in the most abundant one, however,

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the activity in psoriasis treatment has not been reported yet (Lin, 1993). Our preliminary data suggest that indigo blue may exert an antiangiogenic effect which may also partially explain the efficacy of indigo naturalis. Indigo blue extracted from some of the indigo-producing plants was reported to possess a hepatoprotective effect (Sreepriya et al., 2001). Indirubin and its analogues have been used to treat psoriasis orally in China (Lin, 1993). In addition, clinical trials showed that indirubin has a definite efficiency against chronic myelogenous leukemia (Han et al., 1988; Xiao et al., 2002). Indirubin and its analogues selectively inhibit CDKs and other kinases and block cell proliferation in the late G 1 and G2/M phase of the cell cycle (Marko et al., 2001). The promotion of neutrophilic differentiation of human myelocytic leukemia HL-60 cells by indirubin has also been reported (Suzuki et al., 2005). Furthermore, recent studies have revealed that indirubin simultaneously targets several signaling/molecules involved in the pathological process of cancer. The targets include those involved with cell cycle regulation (Hoessel et al., 1999), growth factor receptor signaling (Stat3) (Nam et al., 2005) and AhR signaling (Knockaert et al., 2004). However, mechanism underlying the efficacy of indirubin in psoriasis is still unclear, and the pharmacology of indirubin in keratinocytes also is unknown. Tryptanthrin, another bioactive ingredient from indigo-producing plants, has been reported to exhibit anti-tumor, anti-inflammatory and antiallergic activities (Lin, 1993). Whether tryptanthrin play any role in the anti-psoriatic effect of indigo naturalis is not elucidated and we will further investigate its pharmacology. SUMMARY

Although the western medicines currently used for psoriasis treatments can produce clinical improvement at controlling the disease, none are universally safe and effective, and each carries a considerable risk profile. The clinical responses are sometimes typically short duration, and may be accompanied by disease rebound after therapy is withdrawn. All these therapies for psoriasis are only remissive, but not curative. Recently, alternative therapy (such as herbal remedies, nutritional supplements, and dietary manipulation) for the treatment of psoriasis has increasingly received much attention (Traub & Marshall, 2007). Most common reason cited for patients with psoriasis who are interested in alternative therapy was the dissatisfaction with their conventional treatment. Chinese herbal medicines have demonstrated their efficacy in treating psoriasis for hundreds of years. Unfortunately, the use of traditional Chinese herbal medicine, a mixture of various herbs individually formulated for the patient makes scientific analysis extremely difficult. The mechanisms of these herbs were unknown and any possible adverse effect associated these herbs has fallen on dead ears. In this article, we attempt to provide evidence-based information on the clinical benefit of using topical indigo naturalis as a

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mono-therapy for psoriasis and explore the potential mechanisms of indigo naturalis in treating psoriasis. In our experience, the use of topical application can avoid first-pass effect and chemical degradation of the drug in the gastrointestinal tract, as well as minimizing the risk of toxic side effects including gastric irritation, and liver damage. As psoriasis is a persistent and recurring disease, patients often require continuous treatment preferably with a low cumulative toxicity potential. The use of topical application not only presents direct and better therapeutic efficacy but also avoids the long-term adverse reactions. The effects for the use of indigo naturalis have been described in Chinese medical terms as "clearing the heat and cooling the blood" (Lin, 1993). However, the mechanisms underlying the effects of indigo naturalis specially for treating psoriasis seem to be very limited in the published literature. Psoriasis has three principal histological features: epidermal hyperplasia; dilated, prominent blood vessels in the dermis; and an inflammatory infiltrate of leucocytes, predominantly into the dermis (Griffiths & Barker, 2007). Since psoriatic plaques are likely to arise from the failure of normal differentiation process, reagents that suppress hyperproliferation and promote differentiation of epidermal keratinocytes, as well as inhibit inflammation may have a significant impact on the treatment of psoriasis. Our pathological findings verified the above hypothesis that indigo naturalis is indeed effective to improve psoriatic lesion by controlling hyperproliferation and abnormal differentiation of epidermal keratinocytes, as well as lymphocyte infiltration. In addition, as shown in our histological findings, indigo naturalis may have the effect of recovering the epidermal barrier function. In our in vitro study, we were able to demonstrate that the proliferation of epidermal keratinocytes was down-regulated and differentiation was up-regulated after indigo naturalis treatment. Further investigation of pharmacological mechanism of indigo naturalis and its active components in treating psoriasis are currently performed in our laboratory. In general, topical use of indigo naturalis is relatively safe compared to other currently used therapies. However, one should be aware that most ofthe herb indigo naturalis sold in the Taiwan area (this can happen in any area) is not prepared from natural plant. The chemically synthesized indigo naturalis barely contains any effective ingredient, stains the skin and clothing more easily, and tends to induce hypersensitivity. In conclusion, indigo naturalis topically used for treating psoriasis is both safe and effective. We are very pleased to share our experience in the clinic and basic studies of psoriasis treatment which may contribute to the development of promising therapeutic agents for psoriasis.

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ACKNOWLEDGEMENTS Our studies were supported by Grants CMRPG33024 and CMRPG350301 from Chang Gung Memorial Hospital, Taiwan.

REFERENCES Bedi, M.K. and Shenefelt, P.D. (2002). Herbal therapy in dermatology. Archives of Dermatology, 138: 232-242. Griffiths, C.E. and Barker, J.N. (2007). Pathogenesis and clinical features of psoriasis. Lancet, 370: 263-271. Han, J. (1988). Traditional Chinese medicine and the search for new antineoplastic drugs. Journal of Ethnopharmacology, 24: 1-17. Han, R. (1994). Highlight on the studies of anticancer drugs derived from plants in China. Stem cells, 12: 53-63. Hoessel, R., Leclerc, S., Endicott, J.A., Nobel, M.E., Lawrie, A., Tunnah, P., Leost M, Damiens, E., Marie, D., Marko, D., Niederberger, E., Tang, W., Eisenbrand, G. and Meijer, L. (1999). Indirubin, the active constituent of a Chinese antileukaemia medicine, inhibits cyclindependent kinases. Nature Cell Biology, 1: 60-67. Knockaert, M., Blondel, M., Bach, S., Leost, M., Elbi, C. and Hager, G.L. (2004). Independent actions on cyclin-dependent kinases and aryl hydrocarbon receptor mediate the antiproliferative effects of indirubins. Oncogene, 23: 4400-4412. Koo, J. and Arain, S. (1998). Traditional Chinese medicine for the treatment of dermatologic disorders. Archieves of Dermatology, 134: 1388-1393. Koo, J. and Desai, R. (2003). Traditional Chinese medicine in dermatology. Dermatologic Therapy, 16: 98-105. Lebwohl, M. (2003). Psoriasis. Lancet, 361: 1197-1204. Lin, X.R. (1993). Psoriasis in China. The Journal of Dermatology, 20: 746-755. Lin, Y.K., Yen, H.R., Wong, W.R., Yang, X.H. and Pang, J.H. (2006). Successful treatment of pediatric psoriasis with indigo naturalis composite ointment. Pediatric Dermatology, 23: 507-510. Lin, Y.K, Wong, W.R. and Pang, J.H. (2007). Successful treatment of recalcitrant psoriasis with indigo naturalis ointment. Clinical and Experimental Dermatology, 32: 99-100. Lin, Y.K, Wong, W.R., Chang, Y.C., Chang, C.J., Tsay, P.K, Chang, S.C. and Pang, J.H. (2007). The efficacy and safety of topically applied indigo naturalis ointment in patients with plaque-type psoriasis. Dermatology, 214: 155-16I. Lin, Y.K, Chang, C.J., Chang, Y.C., Wong, W.R., Chang, S.C. and Pang, J.H. (2008). Clinical assessment of patients with recalcitrant psoriasis in a randimized, observer-blind, vehiclecontrolled trial using indigo naturalis. Archives of Dermatology, 144: 1457-1464. Lin, Y.K., Leu, Y.L., Yang, S.H., Chen, H.W., Wang, C.T. and Pang, J.H. (2009). Antipsoriatic effects of indigo naturlis on the proliferation and differentiation of Kerotinozytes with indirubin as the active component. Journal of Dermatoloued Science, 54: 168-174. Marko, D., Schatzle, S., Friedel, A., Genzlinger, A., Zankle, H., Meijer, L. and Eisenbrand, G. (2001). Inhibition of cyclin-dependent kinasel (CDKl) by indirubin derivatives in human tumor cells. Brit~sh Journal of Cancer, 84: 283-289. Nam, S., Buettner, R., Turkson, J., Kim, D., Cheng, J.Q., Muehlbeyer, S., Hippe, F., Vatter, S., Merz, KH., Eisenbrand, G. and Jove, R. (2005). Indirubin derivatives inhibit State signaling and induce apoptosis in human cancer cells. Proceedings of the National Academy of Sciences to the United States of Aermica, 102: 5998-6003. Ortonne, J.P. (1996). Aetiology and pathogenesis of psoriasis. The British Journal of Dermatology, 135: 1-5. Sreepriya, M., Devaki, T. and Nayeem, M. (2001). Protective effects of Indigofera tinctoria L. against D-Galactosamine and carbon tetrachloride challenge on 'in situ' perfused rat liver. Indian Journal of Physiology and Pharmacology, 45: 428-434.

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Suzuki, K, Adachi, R., Hirayama, A., Watanabe, H., Otani, S., Watanabe, Y. and Kasahara, T. (2005). Indirubin, a Chinese anti-leukaemia drug, promotes neutrophilic differentiation of human myelocytic leukaemia HL-60 cells. British Journal Haematology, 130: 681-690. Traub, M. and Marshall, K (2007). Psoriasis-pathophysiology, conventional, and alternative approaches to treatment. Alternative Medicine ReUlew, 12: 319-330. Tse, T.W. (2003). Use of common Chinese herbs in the treatment of psoriasis. Clinical and Experimental Dermatology, 28: 469-75. Verucchi, G., Calza, L., Attard, L. and Chiodo, F. (2002). Acute hepatitis induced by traditional Chinese herbs used in the treatment of psoriasis. Journal of Gastroenterology and Hepatology, 17: 1342-1343. Xiao, Z., Hao, Y., Liu, B. and Qian, L. (2002). Indirubin and meisoindigo in the treatment of chronic myelogenous leukemia in China. Leukemia & Lymphoma, 43: 1763-17688. Yip, S.Y. (1984). The prevalence of psoriasis in the Mongoloid race. Journal of the American Academy of Dermatology, 10: 965-968. Yuan, Z.Z., Yuan, X. and Xu, Z.X. (1982). Studies on tabellae indigo naturalis in treatment of psoriasis. Journal of Traditional Chinese Medicine, 2: 306.

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3 Development of Anti-Angiogenesis Therapies for Treating Cancer from Chinese Medicinal Herbs STEPHEN

M. SAGAR l ,*, RAlMOND K. WONG l

AND DONALD R.YANCE JR.2

ABSTRACT A new pharmacological approach for managing cancer should simultaneously target the multiple biochemical and physiologic pathways that promote cancer progression. Complex mixtures ofphytochemicals used at relatively low doses can reduce tumor adaptation and resistance to conventional cytotoxic chemotherapy, as well as the prevention ofprogression of microscopic disease following definitive therapy. Angiogenesis is a key process in the promotion of cancer. Many plant-derived chemical mixtures manifest both antiangiogenic as well as other anticancer activities. The present article focuses on products that have a high degree of antiangiogenic activity, but it also briefly describes some of the many other actions of these agents that can inhibit tumor progression and reduce the risk of metastases. Natural health products target molecular pathways other than angiogenesis, including epidermal growth factor receptor, the HER2/neu gene, the cyclooxygenase- 2 enzyme, the nuclear factor kappa-B transcription factor, the protein kinases, the Bcl-2 protein, and coagulation pathways. Antiangiogenic Chinese medicinal herbs include Artemisia annua (Chinese wormwood), Curcuma longa (curcumin), Scutellaria baicalensis (Chinese skullcap), Magnolia officinalis (Chinese magnolia tree), Camellia sinensis (green tea), Ginkgo biloba, Poria cocos, Zingiber officinalis (ginger), Panax ginseng, and Rabdosia rubescens hora (Rabdosia). Quality assurance of appropriate extracts is essential prior to embarking upon clinical trials. More data are required on dose-response, appropriate combinations, and 1. Associate Professor, Departments of Oncology and Medicine, McMaster University,

Canada. 2. Center for Natural Healing, Ashland, Oregon. * Corresponding author: E-mail: stephen.saga~cc.hhsc.ca.

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potential toxicities. Given the multiple effects of these agents, their future use for cancer therapy probably lies in synergistic combinations. During active cancer therapy, they should generally be evaluated in combination with chemotherapy and radiation. In this role, they act as modifiers of biologic response or as adaptogens, potentially enhancing the efficacy of the conventional therapies. Key words : Angiogenesis, anti angiogenic, natural health products, herbal medicine, anticancer, molecular biology

INTRODUCTION The Importance of Antiangiogenesis Therapies for Preventing Cancer Progression The induction and promotion of cancer is a multistep process that involves biochemical interactions from the level of the genes, through cell-signaling pathways, intercellular communication mechanisms, supply of nutrients, channels for metastases, and a host immune response. Part of this process is the induction of new blood vessels to supply nutrients and immunocytes. Many natural health products that inhibit angiogenesis also manifest other anticancer activities. This multi-targeted approach may have major advantages for the treatment of cancer by overcoming the therapeutic resistance to the single drug approach caused by multiple redundant physiological pathways in the cancer cell. Cancer progression requires a source of nutrition and oxygen. Tumors that outgrow their oxygen supply cannot form masses more than 1 to 2 mm in size or they then develop central necrosis. Neoplasms are genetically plastic and often adapt by switching on genes that result in an increased ability to invade and to metastasize. A critical part of this process is the induction of local small blood vessels, termed angiogenesis (Fidler, 2000a; Fidler, 2000b). Tumors do not grow progressively unless they induce a blood supply from the surrounding stroma. Cancers that lack angiogenesis remain dormant. Rapid logarithmic growth follows the acquisition of a blood supply. The tumor angiogenic switch is activated (Fig 1) when the balance of angiogenic inhibitors to stimulators is shifted (Wallace, 2002). The process of neovascularization is subtly controlled in normal tissues by a series of endogenous polypeptides that are secreted during growth, healing, and tissue renewal (Table 1). Cancers synthesize or induce some of these polypeptides, especially vascular endothelial growth factor (VEGF) and angiopoietin (APN). These are peptides that are stimulated by hypoxia and result in sprouting of endothelial cords. The cancer induces a profuse but immature network of thin endothelial-lined channels, essential for tumor oxygenation. Although these new vessels allow progressive tumor growth, they are less efficient than the vascular supply of normal tissues.

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APN normally recruits pericytes and initiates modeling of the vessel wall to more mature forms. However, tumors secrete a relative excess ofVEGF, and this results in disorganized and leaky vessels that cause local bleeding and edema. Antiangiogenic therapy is less susceptible to the development of treatment resistance because it is directed to stromal tissue rather than the genomically unstable tumor cells. Table 1. Endogenous angiogenic polypeptides • • • • • • • • • • • • • • • • • • •

Angiogenin (AG) and Angiotropin (AT) Basic Fibroblast Growth Factor (bFGF) Granulocyte-Colony-Stimulating Factor (G-CSF) Hepatocyte Growth Factor (HGF) Interleukin-8 (11-8) Placental Growth Factor (PGF) Platelet-Derived Endothelial Cell Growth Factor (PD-ECGF) Pleiotrophin (PTN) Proliferin Transforming Growth Factor-a (TGF-a) Transforming Growth Factor-13 (TGF-13) Tumour Necrosis Factor-a (TNF-a) Vascular Endothelial Growth Factor (VEGF) Vascular Permeability Factor (VPF) Insulin-Like Growth Factor I and II (IGF-l and II) Cyclooxygenase (COX) and Lipoxygenase (LOX) Nuclear Factor-Kappa 13 (NF-KB) Activator Protein (AP-l) Angiopoietin (APN)

• Tumour angiogenic switch is activated when balance of angiogenic inhibitors to stimulators is shifted • Reduced production of thrombospondin (TSP-l) from fibroblasts • Vascular endothelial factor (VEGF) stimulates new vessel formation but exceeds Angiopoietin (APN) that normally induces differentiation (pericytes) • Matrix metalloproteinase induction • Increased VEGF relative to APN results in disorganized leaky vessels • Rapid logarithmic growth follows the acquisition of a blood supply

Fig 1. The angiogenic switch

Single antiangiogenic agents, such as monoclonal antibodies or specific tyrosine kinase inhibitors, seem to have limited efficacy. Natural health products contain a range of complex organic chemicals that have synergistic activity. They inhibit angiogenesis by interacting with multiple pathways, as well as having other activities that can interact with cell signaling, the apoptotic pathway, and the interaction of cancer cells with the immune system. Some antiangiogenic agents also have anticoagulation activity that may also be associated with a reduction of metastases. Heparin is a well known example of a therapy with both anticoagulation and antiangiogenic activities. Instead of developing multiple monoclonal antibodies to target

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the various peptides and their receptors, an alternative approach would be to evaluate phytochemicals that influence multiple pathways. The science of pharmacognosy evaluates drugs derived from herbal remedies or phytomedicines. There has been minimal clinical research that evaluates their use as adjuvant therapy to conventional treatment with cytotoxic drugs and radiotherapy. Antiangiogenic natural health products may be most effective in impeding cancer recurrence after cytotoxic therapy, encouraging tumors to remain dormant by changing the balance from cell proliferation to cell death by apoptosis.

The Angiogenic-Metastatic Pathway as a Target for Anticancer Therapies The process of cancer metastasis consists of a series of sequential interrelated steps. Each step is rate limited and may be a target for therapy. The outcome of the process is dependent on both the intrinsic properties of the tumor cells and the responses of the host. The balance of these interactions varies between tumors and patients. The major steps in the formation of a metastasis are as follows (Sugarbaker, 1979; Hart et al., 1989; Liotta & Stetler-Stevenson, 1991): 1. Transformation of normal cells into tumor cells followed by growth. Initially depends on nutrients supplied by simple diffusion. 2. Extensive vascularization (angiogenesis). This must occur if the tumor mass is to exceed 1 mm in diameter. The production and secretion of proangiogenic factors by tumor cells and host cells play a major role in establishing a capillary network from the surrounding host tissue. 3. Local invasion. Tumor cells invade the host stroma through several mechanisms. Thin-walled venules, fragmented arterioles, and lymphatic channels offer little resistance to penetration and provide the most common pathways for tumor cell entry into the circulation. 4. Detachment and embolization. Single cells or clumps break away. Most circulating tumor cells are rapidly destroyed. Those that survive must arrest in the capillary beds of distant organs by adhering either to capillary endothelial cells or to the exposed subendothelial basement membrane. 5. Extravasation into new host organ or tissue. 6. Proliferation within the new host organ or tissue. To continue growing beyond the size of 1 mm in diameter, the micrometastasis must develop a vascular network and evade destruction by host defenses. The cells can then continue to invade blood vessels, enter the circulation, and produce additional metastases. The growth of many cancers is associated with the absence of the endogenous inhibitors of angiogenesis, such as interferon-beta (INF -~). INF-

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~ is a potent inhibitor of angiogenesis through blocking interleukin-8 (IL8), basic fibroblast growth factor (bFGF), and collagenase type V, which are all potent angiogenic factors that aid tumor development and invasiveness. VEGF stimulates the proliferation and migration of endothelial cells and induces the expression ofmetalloproteinases and plasminogen activity. Over expression of VEGF in tumor cells enhances tumor growth and metastasis in several animal models by stimulating vascularization (Folkman & Kotran, 1976; Senger et al., 1983; Folkman et al., 1986; Folkman & Klagsbrun, 1987; Nagy et al., 1989; Folkman et al., 1995; Ferrera et al., 1996; Risau, 1997; Kumar et al., 1998). Some cytotoxic chemotherapy agents are being used at lower than normal doses with the intent of inhibiting angiogenesis and minimizing toxicity (Oliff et al., 1996; Miller et al., 2001). This strategy may permit advanced cancer patients to maintain a better quality of life. The low-dose therapy is termed metronomic dosing (Hanahan et al., 2000; Maraveyas et al., 2005; Hudis, 2005). The metronomic model of conventional cytotoxic chemotherapy suggests that there may also be advantages for administering combinations of phytochemicals that interact with the multistep process of angiogenesis (Singh et al., 2003). In other words, targeting the vascular endothelium with continuous low-dose non-cytotoxic therapies may maintain tumor control without excessive toxicity. Their potential role for increasing overall survival (but not necessarily diseasefree survival) and maintaining quality of life requires evaluation in future clinical trials.

Screening Herbs for Antiangiogenic Activity One ofthe first isolated antiangiogenic agents was a phytochemical. Ingber et al. (1990) reported the antiangiogenic properties of fumagillin, a secreted antibiotic of the fungus Aspergillus fumigatus (Trichocomaceae). Refined fumagillin produces excess toxicity, so analogues of fumagillin were subsequently synthesized. Fumagillin and an analogue labeled TNP-470 are proposed to inhibit angiogenesis by selective inhibition of methionine aminopeptidase type 2 (MetAP-2). However, TNP-470 also demonstrated poor pharmacokinetic behavior and dose-limiting toxicity in clinical trials, and these factors remain obstacles to its use as an anticancer agent. Further modifications of fumagillin have been conducted to develop MetAP-2 inhibitors with desirable pharmacological properties. They have been tested only by in vitro assays, and to date, no clinical trials of these analogues have yet been conducted (Ingber et al., 1990; Pyun et al., 2004; Furness et al., 2005). Since the angiogenic cascade is a multistep process, numerous assays have been developed to study potential angiogenic activity. Some analyze a single step in the pathway, whereas others test the angiogenic cascade as a whole. The relationship of each assay to clinical activity is poorly defined. Some agents have profound anti angiogenic effects at low doses; others exhibit anti angiogenic activity only at near cytotoxic concentrations. Some agents have activity in one model but none in others.

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Criteria for anti angiogenic activity are listed in Fig 4 and should include (Miller et al., 2001): • differential cytotoxicity, • alteration of endothelial cell function, • critical mechanistic effects, and • inhibition of angiogenesis in vivo. Western Biomedicine

Traditional Chinese Medicine • Stagnation of blood and Qi

• Destagnation herbs

• Increased fluid content and abnormal vascular supply; blood stasis; poor oxygenation • Anticoagulants as adjunct to chemotherapy increase survival in clinical studies; prevent blood-borne metastases in animal models

Fig 2. The traditional chinese medicine versus the biomedical theory of cancer

Various assays (Table 2) are used to screen natural health products for antiangiogenic activity (Miller et al., 2001; Wang et al., 2004; Kruger et al., 2001). Assays used for screening will be briefly discussed. Table 2. Screening for anti-angiogenesis

• In vitro Assays - Human umbilical vein endothelial cells (HUVEC) - Bovine aortic endothelial cells (BAEC) - Tumour endothelial cells (TEC) • In vivo Assays - Chick embryo chorioallantoic membrane (CAM) model - Cornea implantation - Matrigel - Tumour xenografts

In vitro Assays In vitro assays are designed to recapitulate each of the multiple events that constitute the angiogenic process. Some of them are very specific in analyzing a single event (proliferation, apoptosis, migration, production of proteases), whereas others provide a more complex picture of the process involving cell functions and interactions with the environment. In vitro assays for the activity of anti angiogenic compounds are usually based on the use of endothelial cells. A critical issue in setting up an in vitro assay is the choice of endothelial cells. Immortalized endothelial cells are sometimes used, as they provide an "unlimited" source of cells. Although these cell lines have the obvious advantages of being easy to grow and relatively stable

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throughout in vitro passages and among batches, they have usually lost some of the characteristics of endothelial cells, including molecular markers, and exhibit changes in function. The most commonly used endothelial cells are from the human umbilical vein, since these are easily available and cell isolation is relatively simple. For the same reasons, bovine or murine aortic endothelial cells are often used too, but these come from large vessels, and they have different phenotypic and behavioral characteristics from those of the microvessels that are more likely involved in angiogenesis. Other common sources of microvascular endothelial cells are the skin, brain, adipose tissue, and adrenal gland. Endothelial cells derived from the microvasculature of different tissues/organs are often heterogeneous, imposing a further constraint on the choice of cell model. Ideally, when developing inhibitors of tumor angiogenesis, tumor-derived endothelial cells should be used. However, practical difficulties in their isolation from tumor tissue and maintenance in culture have limited their use in preclinical studies (Taraboletti et al., 2004). The ability to maintain endothelial cells in culture has allowed the study of endothelial cell proliferation, migration, and cellular function. Angiogenic activity may be represented as endothelial cell migration across a Boyden chamber. Compounds with antiangiogenic potential will inhibit the migration. The bovine aortic endothelial cell (BAEC) and the human umbilical vein endothelial cell (HUVEC) assays are established systems. In vitro assays are relatively inexpensive and give more rapid results. However, the ability to inhibit endothelial cell proliferation, migration, and tubule formation in vitro may not necessarily predict in vivo response. In vitro assays are a rapid method for initial screening of large numbers of agents. Definitive conclusions cannot be based on in vitro assays alone.

In vivo Assays These biological assays are more specific for detecting antiangiogenic activity. The chick embryo chorioallantoic membrane (CAM) model is an extraembryonic membrane that is commonly used to study agents that influence angiogenesis. An angiogenic response occurs 72 to 96 h after stimulation in the form of increased vessel density around the implant. On the other hand, an angiostatic compound induces the vessels to become less dense around the implant and even disappear. Other systems include animal cornea implantation, disc angiogenesis, Matrigel systems, and tumor xenograft models. The in vivo assays provide a more complete physiologic assessment of angiogenesis but are more time-consuming and expensive.

Criteria for Antiangiogenic Activity The degree of anti angiogenic activity is dose dependent. Most chemotherapy drugs have antiangiogenic activity when administered at high doses. We

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are especially interested in compounds that specifically interact and antagonize the steps involved in angiogenesis when administered at low doses. These agents may have relatively low toxicity at low dose and may exhibit a higher therapeutic gain. Most conventional chemotherapy drugs have some degree of anti angiogenic activity as a consequence of their cytotoxic activity. Ideal botanical derivatives would specifically antagonize new vessel formation in tumors, without significant toxicity to normal tissues and without major adverse reactions. The ideal agent would also inhibit tumor cell proliferation through other physiologic pathways, such as influencing intracellular signaling pathways. Multiple levels of antiangiogenic activity may be required to overcome the development of resistance by tumor-associated endothelial cells (TEC). Survival factors, such as the increased secretion of VEGF and bFGF by the tumor cells, activate intracellular pathways that prevent TEC apoptosis. Maximal anti angiogenic activity usually requires prolonged exposure to low concentrations ofthe active agent. This approach contrasts with the concept of administering maximum tolerated doses of cytotoxic drugs to maximize tumor cell kill. Some reports have confirmed the utility of combining low, frequent-dose chemotherapy plus an agent that specifically targets the endothelial cell compartment (Hanahan et al., 2000; Maraveyas et al., 2005). The evidence suggests that an anti angiogenic schedule can be more effective than using high-dose cytotoxic drugs alone. We hypothesize that concomitant scheduling of antiangiogenic botanicals with low, frequentdose cytotoxic therapies may have biological advantages that can increase therapeutic gain.

Natural Health Products that Inhibit Angiogenesis Further research is necessary to screen herbs that may be useful anti angiogenic therapies. Table 3 lists Chinese herbs with anti angiogenic activity (Wang et al., 2004), and Table 4 lists herbs and their derivatives that inhibit VEGF (Singh et al., 2003). An expert herbalist can advise on potential herbal treatments derived from advanced traditional medical systems, such as traditional Chinese medicine. It will be imperative to develop a new model of modern pharmacology based on traditional pharmacognosy. Our developing knowledge of cancer biology suggests that administering cytotoxic drug therapy at very high doses is not always appropriate. A new approach is to administer lower doses of synergistic organic chemicals. These complexes already exist in myriad botanicals. New laboratory techniques allow more specific assays of activity and enable quality assurance and consistency between batches of botanical preparations to be maintained. This will enable credible clinical trials of antiangiogenic natural health products to be initiated.

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Table 3. Antiangiogenesis activity of chinese medicinal herbal extracts (Exhibiting more than 20% inhibition at 0.2 g/herb/mL) (Wang et al., 2004) Assays: chick embryo chorioallantoic membrane (CAM) and bovine aortic endothelial cells culture models (BAEC) Used Part

Name

Berberis paraspecta Catharanthus roseus Coptis chinensis Scrophularia ningpoensis Scutellaria baicalensis Polygonum cuspidatum Taxus chinensis

% Inhibition

% Inhibition

(CAM)

(BAEC)

25 27 25 20 27

38 30 37 34 41 28

Root Leaf Rhizome Root Root Whole plant Bark

26

Traditional Chinese Medical (TCM) theory describes tumor formation as stagnation of qi and blood (Fig 2). In Western theory, this corresponds to the abnormal vasculature induced by the cancer when it induces the angiogenic switch. The tumor has a stagnant blood supply with abnormal leaky vessels that raises the interstitial fluid pressure, resulting in centrifugal diffusion and poor oxygenation. TCM theory suggests "destagnation herbs" as a potential therapy. A randomized placebocontrolled trial from China showed that the addition of "de stagnation" herbs (including Salvia miltiorrhiza Bunge [Lamiaceael and Angelica sinensis Diels [Apiaceae]) to radiotherapy doubled both the local control and survival rates of patients with nasopharyngeal cancer (Fig 3) (Xu et al., 1989). In some clinical trials, anticoagulation drugs are associated with a reduction in metastases (Hejna et al., 1999; Smorenburg et al., 2001; Blom et al., 2005). Laboratory evidence now suggests that many of these Chinese herbs have anti angiogenic and anticoagulation properties (Huang et al., 2003; Wang et al., 2004; Samuels, 2005).

Destagnation herbs (Huo Xue Hua Yu) activate blood flow and improve micro-circulation (huang qi, chi shao, chuan xiong, dang gui, tao ren, hong hua, ji xue teng, ge gen, chen pi, dan shen) N XRT alone XRT + herbs

90 98

Local recurrence

5 Year survival

29% 14% (p<0.05)

37% 53% (p<0.05)

Fig 3. A randomized controlled trial of chinese herb de stagnation formula with radiation in the treatment of nasopharyngeal carcinoma (Xu et al., 1989)

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Some herbs that are traditionally used in China as anticancer agents have been screened for their antiangiogenic activity (Fig 4) in the laboratory (Wang et ai., 2004). Table 3 lists the most active herbs (exhibiting more than 20% inhibition at 0.2 g/herb/mL), using the CAM and BAEC assays. Other herbs are included in Table 4 and are discussed in more detail. Table 4. Chinese herbs and their derivatives that specifically inhibit vascular endothelial growth factor and have direct activity against angiogenesis

• Artemisia annua (Chinese wormwood; contains 95% artemisinin and other related terpenes and flavonoids) • Scutellaria baicalensis (Chinese Baical skullcap; contains 95% baicalin and flavonoids) • Curcuma Zonga (turmeric; contains 95% curcumin) • Camellia sinensis (green tea; contains 95% phenols; 50% epigallocatechin) • Angelica sinensis (Dong quai; contains 4-hydroxyderricin) • Taxus brevifolia (Pacific yew; contains taxol) • Polygonum cuspidatum (Japanese knotweed; contains 20% resveratrol) • Silybum marianum (Milk thistle; contains 80% silymarin [silibin]) • Ginkgo biloba • Magnolia obovata (contains 90% honokiol) • Zingiber officinale (contains 6-gingerol) • Poria cocus • Panax ginseng • Rabdosia rubescens (Rabdosia) • Berberis paraspecta Ahrendt (Berberidaceae), root • Catharanthus roseus G Don (Apocynaceae), leaf • Coptis chinensis Franch (Ranunculaceae), rhizome • Scrophularia ningpoensis Hemsl (Scrophulariaceae), root • Polygonum cuspidatum, Whole plant • Taxus chinensis Rehder (Taxaceae) • Degree of antiangiogenic activity is dose dependent • Specifically interacts and antagonizes angiogenesis at low doses (high therapeutic gain) • Inhibits tumor cell proliferation through multiple physiologic routes, such as intracellular signaling pathways • Minimal effect on normal angiogenesis • Low toxicity with prolonged administration Fig 4. Criteria for an anti-angiogenic botanical

Botanicals usually act on multiple anticancer targets since they contain a variety of organic chemical complexes. The biochemical signaling pathways of angiogenesis form a complex, interconnected web. Inhibition of one part of this web may result in compensation through another pathway. A potential advantage of phytochemicals is that they may act through multiple pathways and reduce the development of resistance by cancer cells. This model of pharmacognosy recognizes the advantage of administering the whole plant product to maximize activity (Fig 5). Over extraction of a specific chemical constituent may remove this therapeutic gain. The

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challenge for modern pharmacognosy is to ensure that the optimum mixture of chemical constituents is maintained when purifying the product. Usually, this will require a combination of both chemical and biological assays. Further anticancer properties of some anti angiogenic botanicals will briefly be discussed. Their effects may also interact with various biochemical pathways that indirectly influence angiogenesis. Traditional practice has been to combine multiple natural health products, and this may scientifically provide a therapeutic advantage. • Range of complex organic chemicals that may have synergistic activity, alone or in combinations. • Inhibit angiogenesis by interacting with multiple pathways. • Bonus activities: interact with cell signaling, apoptosis and interaction of cancer cells with the immune system. • Anticoagulation activity that may be associated with a reduction of metastases • Less acquired resistance to therapy. • Low toxicity: suitable for combination therapy and long term administration. • Possible economic advantages. Fig 5. Advantages of anti-angiogenic NHPs

Chinese Medicinal Botanicals for Potential Development into AntiAngiogenic Therapeutic Agents for the Treatment of Cancer

Artemisia annua (Chinese Wormwood) Artemisinin is the active constituent extracted from the plant A. annua L. (Asteraceae). It has been used clinically as an antimalarial drug (Mueller et al., 2004). More recently, it was shown to be cytotoxic to cancer cells through induction of apoptosis (Singh & Lai, 2004). Artesunate (ART) is a semisynthetic derivative of artemisinin. The in vitro effect of ART was tested on the HUVEC model of angiogenesis. It significantly inhibited angiogenesis in a dose-dependent manner. The inhibition of HUVEC proliferation was greater than the effect on cancer cells, fibroblast cells, and human endometrial cells. This indicates that its anti angiogenic activity is greater than its cytotoxicity. The antiangiogenic effect in vivo was evaluated in nude mice using transplanted human ovarian cancer (HO891) cells and immunohistochemical staining for microvessel CD31 antigen, VEGF, and the VEGF receptor (KDRlflk-1). Tumor growth was decreased and microvessel density was reduced without any toxicity to the host animals. Artemisinin also lowered the VEGF expression by tumor cells and the KDRlflk-1 expression by endothelial cells (Chen & Chang, 2004). Artemisinin also has therapeutic effects through other pathways. It inhibits the activation of nuclear factor-KB (NF-KB), an important activator protein in cancer development and progression (Aldieri et al., 2003).

Scutellaria baicalensis (Chinese Skullcap) Baicalin and baicalein are the main derivatives from the Chinese Skullcap herb, S. baicalensis Georgi (Lamiaceae). They are potent anti angiogenic

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compounds that reduce VEGF, bFGF, 12-lipoxygenase activity, and MMP (Liu et aZ., 2003; Miocinovic et aZ., 2005). It is a component of the traditional Bupleurum formula for stagnation (Xiao Chai Hu Tang): • Bupleurum (Chai Hu) 12-15 g BupZeurum chinense • Scutellaria (Huang Qin) 9-12 g Scutellaria baicaZensis • Pinelli a (Ban Xia) 9-12 g Pinellia ternata • Fresh Ginger (Sheng Jiang) 3-6 g Zingiberis offici naZis • Ginseng (Ren Shen) 6-9 g Panax ginseng • Licorice (Gan Cao) 3-6 g GZycyrrhiza uraZensis • Jujube Dates (Da Zao) 3-5 pcs Zizyphus jujuba S. baicaZensis is also one of the herbs found in PC-SPES, a complex of Chinese herbs that may have clinical activity against advanced prostate cancer (Small et aZ., 2000; Oh et aZ., 2001). However, although a phase 2 trial demonstrated clinical activity in patients with androgen independent prostate cancer, diethylstilbestrol (DES) and ethinyl estradiol (both known to have potent antiprostate cancer activity) were detected in various lots of PC-SPES (Hsieh et aZ., 2002). It is still intriguing that the decline in PSA was greater for PC-SPES that was potentially contaminated with DES than for the comparator group that received DES alone, suggesting some independent activity. Although baicalin and baicalein have multiple anticancer activities in vitro, their clinical activity is not established, and their contribution to any potential therapeutic effect of PC-SPES is unknown (Cordell, 2002; Oh et aZ., 2004).

Curcuma longa (Turmeric) Curcumin is the most active curcuminoid present in turmeric, C. Zonga L. (Zingiberaceae). It interacts with cancer cells at a number of levels and can enhance the tumoricidal efficacy of cytotoxic chemotherapy and radiotherapy (Narayan, 2004; Khafif et aZ., 2005; Sen et aZ., 2005). Its antiinvasive effects are partly mediated through the down regulation of MMP2 and the upregulation of tissue inhibitor of metalloproteinase (TIMP-l) (Shao et aZ., 2002). These enzymes are involved in the regulation of tumor cell invasion. Curcumin inhibits the transcription of 2 major angiogenesis factors, VEGF and bFGF (Arbiser et aZ., 1998). It interacts with VEGF and nitric oxide- mediated angiogenesis in tumors (Sreejayan, 1997; GarciaCardena et aZ., 1998). Elevated levels of nitric oxide correlate with tumor growth. Curcumin reduces nitric oxide generation in endothelial cells. CD13/ aminopeptidase-N (APN) is a membrane bound enzyme found in blood vessels undergoing active angiogenesis. Curcumin binds to APN and blocks its activity, thereby inhibiting angiogenesis and tumor cell invasion (Gururaj et aZ., 2002; Shim et aZ., 2003). Derivatives of curcumin may be

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developed to target APN, providing a novel approach to reduce neoplastic angiogenesis (John et al., 2002; Hahm et al., 2004). Curcumin also downregulates the expression of the VEGF and MMP-9 genes that are associated with angiogenesis. Demethoxycurcumin is a structural analogue of curcumin isolated from Curcuma aromatica Salisb. (Zingiberaceae). It specifically inhibits the expression of MMP-9 (Kim et al., 2002). Curcumin can interfere with the activity of both MMP-2 and MMP-9, the basis of the angiogenic switch, thereby reducing the degradation of the extracellular matrix (Chen et al., 2004). It also interferes with the release of angiogenic factors that are stored in the extracellular matrix. It inhibits growth factor receptors such as epidermal growth factor receptor (EGFR) and VEGFR and the intracellular signaling tyrosine kinases. This cell-signaling system can promote further angiogenesis through gene activation that increases levels of cyclooxygenase-2 (COX-2), VEGF, IL-8, and the MMPs (Reddy & Aggarwal, 1994; Dorai et al., 2001; Leu et al., 2003). A phase 1 study of curcumin found no treatment-related toxicity up to 8000 mg/d. Beyond 8000 mg/d, the bulky volume of the drug was unacceptable to the patients. The serum concentration of curcumin usually peaked at 1 to 2 h after oral intake of curcumin and gradually declined within 12 h (Cheng et al., 2001). This study suggested that it may prevent cancer progression. Derivatives of curcumin, such as copper chelates of curcuminoids, may have increased antitumor activity (John et al., 2002).

Camellia sinensis (Green Tea) C. sinensis (L.) Kuntze (Theaceae), contains polyphenols and catechins (mainly epigallocatechin-3 gallate [EGCG]) (Lee et al., 2002). These constituents inhibit MDAMB breast cancer cell and HUVEC proliferation (Sartippour et al., 2002). In addition, they suppress breast cancer xenograft growth and reduce the density of tumor vessels in rodent studies (Cao & Cao, 1999). This is associated with a decrease in VEGF, regulated at the level of transcription. EGCG also suppresses protein kinase C (PKC), another VEGF transcription modulator. Inhibition of VEGF transcription is one of the molecular mechanisms involved in the anti angiogenic effects of green tea that may contribute to its potential use for cancer treatment (Kojima-Yuasa et al., 2003; Tang et al., 2003; Fassina et al., 2004). EGCG may be administered as a powdered extract of green tea. An appropriate dose has been extrapolated from antiangiogenic activity in rodent experiments as well as a phase 1 study in humans (Cao et al., 1999; Pisters et al., 2001). A dose of 1.0 g/m3 times daily (equivalent to 7-8 Japanese cups [120 mLl 3 times daily) has been recommended. In practice, lower total daily doses of 2 to 4 g of standardized green tea extract (95% polyphenols/60% catechins) are usually prescribed. Each gram of this extract provides 400 to 500 mg of EGCG. The dose-limiting adverse effects are gastrointestinal and

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neurological effects of caffeine. However, the caffeine may potentiate the antiangiogenic effect of EGCG (Pisters et al., 2001).

Polygonum cuspidatum (Japanese Knotweed) This contains 20% resveratrol. It has anti angiogenic activity demonstrated by its ability to inhibit HUVEC division and to decrease the lytic activity of MMP-2 (Cao et al., 2005). Resveratrol inhibits VEGF induced angiogenesis by disruption of reactive oxygen species-dependent Src kinase activation and subsequent VE-cadherin tyrosine phosphorylation (Igura et al., 2001; Lin et al., 2003). Resveratrol inhibits the growth of gliomas in rats by suppressing angiogenesis (Tseng et al., 2004).

Silybum marianum (Milk Thistle) Silibinin and silymarin are polyphenolic flavonoids isolated from the fruits or seeds of S. marianum (L.) Gaertn. (Asteraceae). In the laboratory, silymarin demonstrates strong activity against a variety of tumors through down regulating VEGF and EGFR (Jiang et al., 2002; Singh & Aggarwal, 2003). Silymarin suppresses VEGF when used as a single agent against human ovarian cancer endothelial cells in vitro (Gallo et al., 2003). A more detailed discussion can be found elsewhere (Sagar, 2007).

Ginkgo biloba G. biloba L. (Ginkgoaceae) extract has anticancer effects that are related to its gene-regulatory and antiangiogenic properties. The G. biloba extract used in most of the research is EGb 761, which contains about 25% flavonoids (ginkgo-flavone glycosides) and about 5% terpenoids (ginkgolides and bilobalides). The most potent flavonoid is ginkgolide B. This extract inhibits angiogenesis by down regulating VEGF (Zhang et al., 2002; De Feudis et al., 2003).

Magnolia officinalis (Chinese Magnolia Tree) The seed cones of M. officinalis Rehder & E.H. Wilson (Magnoliaceae) contain substances that inhibit the growth of new blood vessels. Honokiol is the active constituent. It may partly reduce angiogenesis through the regulation of platelet-derived endothelial cell growth factor and TGF-f3 expression. It also inhibits nitric oxide synthesis and TNF-f3 expression (Son et al., 2000; Lee et al., 2004). In animal experiments, it suppresses the proliferation of blood vessel endothelial cells more than other types of cells and thereby reduces tumor growth (Bai et al., 2000; Chen et al., 2004).

Zingiber officinale (Ginger) 6-Gingerol, fromZ. officinale Roscoe (Zingiberaceae), inhibits both the VEGFand bFGF-induced proliferation of human endothelial cells and causes cell cycle arrest. It also blocks capillary-like tube formation by endothelial cells in response to VEGF and strongly inhibits sprouting of endothelial cells in

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the rat aorta and mouse cornea in vitro models. In mice receiving injections of B16F10 melanoma cells, intraperitoneal administration of 6-gingerol, at doses less than cytotoxic levels, reduces the number of lung metastases (Kim et al., 2005).

Poria cocos P. cocos F.A. Wolff (Coriolaceae) is a mushroom extract that has been traditionally held to have anticancer activity. It inhibits platelet aggregation

and appears to be antiangiogenic by downregulating NF-kappa-B (Jin et al., 2003; Chen et al., 2004; Lee et al., 2004).

Panax ginseng The lipophilic constituents of P. ginseng C.A. Meyer (Araliaceae) are called saponins (or ginsenosides). These extracts possess anticancer activities in tumors that include antiangiogenesis and induction of tumor cell apoptosis (Sato et al., 1994).

Rabdosia rubescens (Rabdosia) The herb R. rubescens H . Hara (Lamiaceae) is used traditionally to treat cancer and is a constituent of the PC-SPES formula. It contains ponicidin and oridonin, two diterpenoids that possess significant antiangiogenic activity (Meade-Tollin et al., 2004).

CONCLUSIONS Angiogenesis involves multiple interdependent processes operating at the molecular level. These include gene expression, signal processing, and enzyme activities. Most antiangiogenic natural health products block new vessel formation at multiple levels. Lack of standardization of screening assays may be an obstacle to defining the most effective products for clinical use. Over extraction of constituents may negate some of the advantages of potential synergy. Mainly preclinical data exist for most of the naturally derived anti angiogenic agents. Most of the studies of anti angiogenic activity are based on in vitro or animal work, which cannot be readily extrapolated to humans. Phase 1 and 2 studies are required to determine their potential to improve cytotoxic therapies. Quality assurance of appropriate extracts is essential prior to embarking on clinical trials (Fig 6). Since anti angiogenic agents are mainly cytostatic in nature, the usual paradigm for anticancer drug development, in which tumor response in phase 2 trials prompts further development, is not always appropriate. More data are required on dose response, appropriate combinations, and potential toxicities. Given the multiple effects of these agents, their future use for cancer therapy probably lies in synergistic combinations. They may

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4·

wi

Traditional Qbs~tvati!:>nS'

Authentication ;lmf;f~oslational research

Dose optimization

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effi l'ldverse taifects

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be evaluated alone for the prevention of cancer recurrence following definitive treatment. To be suitable for long-term chronic use, these agents should possess minimal toxicity and should be orally administered. However, angiogenesis is also essential for healing of injuries. Most compounds that inhibit tumor angiogenesis are likely to inhibit physiologic angiogenesis, leading to potential side effects, such as ulceration and bleeding. Studies are required to determine distinguishing features of tumor vessels from normal vessels to enable a therapeutic gain to be achieved. During active cancer therapy, they should generally be evaluated in combination with chemotherapy and radiation. In this role, they act as biological response modifiers and adaptogens, potentially enhancing the efficacy of the so-called conventional therapies. The diversity of angiogenic factor expression in different tumors receiving various therapies, combined with the fact that endothelial cells in different tumors are phenotypically distinct, is a major challenge for the development of effective antiangiogenic regimens (Jung et at., 2000; Sweeney et at., 2003). Their effectiveness may be increased when multiple agents are used in optimal combinations. Surrogate markers, such as angiogenic cytokines, are necessary to predict anti angiogenic response. Circulating levels of FGF-2, VEGF, vascular adhesion molecule (V-CAM-1), endothelial intercellular adhesion molecule (ICAM-l), IGF-1, and cytokines such as IL-8 may correlate with tumor angiogenesis (Yoshida et at., 1997; Tang et at., 2001; Brower, 2003; Ruegg, et at., 2003; Ria et ai., 2004). In addition, circulating endothelial cells and their progenitors may be a more reliable marker of response to anti angiogenic therapies (Salcedo et at., 2005; Schneider et ai., 2005; Shaked et ai., 2005). Noninvasive functional imaging, such as positron emission tomography and functional magnetic resonance imaging, may playa role (Neeman, 2000).

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Current laboratory evidence suggests a useful role for natural health products in the treatment of cancer. The input of an ethnobotanist, oncologist, laboratory scientist, and a clinical trials methodologist to the research effort is essential to distill the wealth of traditional knowledge into a modern framework that can be evaluated scientifically. The determination of the biologically active dose that may possess least toxicity may be relevant. Combinations of whole herbs or constituent phytochemicals at lower doses are important. In addition, a longer period of exposure to the natural health product may be more effective than a short exposure to the highest possible dose level. New designs for trials to demonstrate activity in human subjects are required. Although controlled trials might be preferred, smaller studies with appropriate end points and surrogate markers for anti angiogenic response could help prioritize agents for the larger resource-intensive phase 3 trials. Because most of the agents are expected to be cytostatic, it is inappropriate to require the standard criteria of measured tumor response. On the other hand, simply confirming stable disease may be misleading. More research on surrogate markers of antiangiogenic response is obviously necessary prior to directing resources to large-scale clinical trials. A multidisciplinary approach by the herbalist and the oncologist is important for implementing and studying natural health products used for the treatment of patients with cancer. We now have a better understanding of their effects at the molecular level. Introducing these interventions into the clinic through appropriate studies will provide more definitive evidence of efficacy and hopefully improve outcome for many cancer patients.

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Miller, KD., Sweeney, C.J. and Sledge, G.W. (2001). Redefining the target: chemotherapeutics as antiantiogenics. J Clin Oncol., 19: 1195-1206. Miocinovic, R, McCabe, N.P. and Keck, RW. (2005). In vivo and in vitro effect of baicalein on human prostate cancer cells. Int J Oncol., 26: 241-246. Mizushina, Y., Akihisa, T. and Ukiya, M. (2004). A novel DNA topoisomerase inhibitor: dehydroebriconic acid; one of the lanostane-type triterpene acids from Poria cocos. Cancer Sci., 95: 354-360. Mueller, M.S., Runyambo, N. and Wagner, I. (2004). Randomized controlled trial of a traditional preparation of Artemisia annua L. (Annual Wormwood) in the treatment of malaria. Trans R Soc Trop Med Hyg., 98: 318-321. Nagy, J.A., Brown, L.F. and Senger, D.R (1989). Pathogenesis of tumor stroma generation: a critical role for leaky blood vessels and fibrin deposition. Bioch~m Biophys Acta, 948: 305-326. Narayan, S. (2004). Curcumin, a multi-functional chemopreventive agent, blocks growth of colon cancer cells by targeting betacatenin-mediated transactivation and cell-cell adhesion pathways. J Mol H~stol., 35: 301-307. Neeman, M. (2000). Preclinical MRI experience in imaging angiogenesis. Cancer Metastasis Rev., 19: 39-43. Oh, W.K, George, D.J. and Hackmann, K (2001). Activity of the herbal combination, PCSPES, in the treatment of patients with androgen-independent prostate cancer. Urology, 57: 122-126. Oh, W.K, Kantoff, P.W. and Weinberg, V. (2004). Prospective, multicenter randomized phase II trial of the herbal supplement: PC-SPES, and diethylstibestrol in patients with androgen- independent prostate cancer. J Clin Oncol., 22: 3705-3712. Oliff, A., Gibbs, J.B. and McCormick, F. (1996). New molecular targets for cancer therapy. Sci Am., 275: 144-149. Pisters, KM.W., Newman, RA. and Coldman, B. (2001). Phase I trial of oral green tea extract in adult patients with solid tumors. J Clin Oncol., 19: 1830-1838. Pyun, H.J., Fardis, M. and Tario, J. (2004). Investigation of novel fumagillin analogues as angiogenesis inhibitors. Bworg Med Chem Lett., 14: 91-94. Reddy, S. and Aggarwal, B.B. (1994). Curcumin is a non-competitive and selective inhibitor of phosphorylase kinase. Fed Eur Bwchem Soc Lett., 341: 19-22. Ria, R, Portaluri, M. and Russo, F. (2004). Serum levels of angiogenic cytokines decrease after antineoplastic radiotherapy. Cancer Lett., 216: 103-107. Risau, W. (1997). Mechanisms of angiogenesis. Nature, 386: 671-674. Ruegg, C., Meuwly, J.-Y. and Driscoll, R. (2003). The quest for surrogate markers of angiogenesis: a paradigm for translational research in tumor angiogenesis and anti angiogenesis trials. Curr Mol Med., 3: 673-691. Sagar, S.M. (2007). Future directions for research on Silybum marianum for cancer patients. Integrat Cancer Ther., 6: 166-173. Salcedo, X., Medina, J. and Sanz-Cameno, P. (2005). Review article: angiogenesis soluble factors as liver disease markers. Al~ment Pharmacol Ther., 22: 23-30. Samuels, N. (2005). Herbal remedies and anticoagulant therapy. Thromb Haemost., 93: 3-7. Sartippour, M.R, Shao, Z.M. and Heber, D. (2002). Green tea inhibits vascular endothelial growth factor (VEGF) induction in human breast cancer cells. J Nutr., 132: 2307-2311. Sato, K, Mochizuki, M. and Saiki, I. (1994). Inhibition of tumor angiogenesis and metastasis by a saponin of Panax ginseng, ginsenoside-Rb2. Bioi Pharm Bull., 17: 635-639. Schneider, M., Tjwa, M. and Carmelie, P. (2005). A surrogate marker to monitor angiogenesis at last. Cancer Cell., 7: 3-4. Sen, S., Sharma, H. and Singh, N. (2005). Curcumin enhances vinorelbine mediated apoptosis in NSCLC cells by the mitochondrial pathway. Biochem Biophys Res Commun., 331: 1245-1252. Senger, D.R., Galli, S.J. and Dvorak, A.M. (1983). Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid. Science, 219: 983-985. Shaked, Y., Bertolini, F. and Man, S. (2005). Genetic heterogeneity of the vasculogenic phenotype parallels angiogenesis: implications for cellular surrogate marker analysis of antiangiogenesis. Cancer Cell, 7: 101-111.

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4 An Anthology of the Studies on the AntiInflammatory, Antinociceptive and Febrifuge Effects of Medicinal Plants in Turkey ESRA KUPELI AKKOL 1 AND ERDEM YESlLADA2

ABSTRACT Inflammatory diseases are among the most common health problems treated with traditional remedies. Therefore it is crucial to evaluate the potential of herbal remedies for the discovery of novel bioactive compounds that might serve as leads for the development of potent drugs. Although several agents are known to treat these types of disorders, prolonged use should be avoided due to severe side effects. Consequently, there is a need to develop safe and new anti-inflammatory agents with minimum side effects. In this context, traditional medicines provide a vast source for the discovery of original drug leads. The present investigation represents a preliminary screening in an ongoing program on plants used in Turkish traditional medicine for the treatment of rheumatism and related inflammatory diseases. In this study, selected plants as the subject of the present study were recorded in the alphabetical order of plant families. Key words : Antinociceptive, anti-inflammatory, bioassay-guided processing, ethnopharmacology, febrifuge, folk remedies, plants, Turkey 1. Department of Pharmacognosy, Faculty of Pharmacy, Gazi University, Etiler 06330, Ankara, Turkey. 2. Department of Pharmacognosy, Faculty of Pharmacy, Yeditepe University, Kayisdagi 34755 Istanbul-Turkey. * Corresponding author: E-mail: [email protected]

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INTRODUCTION Acute and chronic inflammations are known to be complicated processes induced by several different classes of chemical mediators, e.g. prostaglandins, leukotrienes and platelet-activating factor, etc. Antiinflammatory agents exert their effect through a spectrum of different modes of action (Samuelsson et al., 1978). However, chronic inflammatory diseases are still one of the major health problems worldwide and nonsteroid anti-inflammatory drugs (NSAIDs) are the most prescribed drugs for treatment of inflammatory diseases. Although the NSAIDs provide the patients with symptomatic relief, they do not modify the pathogenesis of inflammation and do not reduce the disabling bone and cartilage damage (Ford-Hutchinson et al., 1981). Therefore, it has become a must to search for new initiatives in the treatment of chronic inflammation. On the other hand, plants have been used by human being since ages in traditional medicine due to their therapeutic potential and the search on medicinal plants have led the discovery of novel drug candidates used against diverse diseases. The plant kingdom encompasses a wide range of plants that possess biologically active molecules of medicinal value. Many such plant-derived molecules have been isolated, identified, and successfully introduced into international markets by pharmaceutical industries that are mostly located in the developed world. In contrast, plant-based remedies, prescribed by folk doctors in the developing world for the treatment of many diseases, remain largely restricted in their use to the local communities in which such remedies were developed (Sumner, 2000). Although such plant-based remedies have recently received considerable interest from the medical and pharmaceutical industries, only a small fraction of such plants have been assessed. In the past few years, many studies have investigated the healing potential of higher plants with ethnobotanical histories. This stemmed from several reasons such as consumer interest in such plantbased remedies, the concern about the possible side effects of allopathic medicine, the lower cost of phytotherapy, and the fact that many plantbased or herbal remedies succeeded in replacing allopathic medicines in relieving malady symptoms. The aim of this manuscript is to review the literature of bioactive anti-inflammatory plant extracts and their isolated active components as an addition to the literature typically used in folk medicine in Turkey basin to treat inflammation related ailments.

ANACARDIACEAE The nuts of Pistacia vera L. (Anacardiaceae), commonly referred to pistachio, is a quite popular flavoring foodstuff and snack in Turkey and in the world. The oleoresin of P. vera, a widely-distributed tree throughout

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the south-east region of Anatolia, has been used to treat asthma by chewing as folk remedy in this part of Turkey as well as against stomachache (by chewing the gum) and hemorrhoids (externally) (Baytop, 1999). In the light oftraditional information, the ethanolic and aqueous extracts prepared from different parts (leaf, branch, fruit, stalk) of Pistacia vera as well as its oleoresin were evaluated for their in vivo anti-inflammatory and antinociceptive activities (Orhan et al., 2006). Only the oleoresin, out of the all extracts screened herein, exhibited a dose-dependent anti-inflammatory activity on carrageenan-induced hind paw edema model in mice ranging between 27.4-34.7% at 250 mg/kg and 32.6-38.7% at 500 mg/kg doses without inducing any gastric damage. The potency of oleoresin at 500 mg/kg dose was almost equal to that observed for indomethacin, a reference agent. As for antinociceptive activity, the oleoresin also displayed 32.1 % inhibition at 500 mg/kg dose onp-benzoquinone induced abdominal contractions in mice, while inhibitory rate decreased to 21.7% at 250 mg/kg. Fractionation of the oleoresin indicated that n-hexane fraction to be active, which further led to recognition of some monoterpenes, mainly a-pinene (77.5%) by capillary gas chromatography-mass spectrometry (GC-MS) as well as the oleoresin itself. a-Pinene was also assessed for its antinociceptive and antiinflammatory activities in the same manner and exerted a moderate antiinflammatory effect at 500 mg/kg dose. APIACEAE In Turkish folk medicine various species of the Eryngium are used for a wide range of ailments; particularly roots are used against various inflammatory disorders, oedema, sinusitis, urinary infections or inflammations etc. and snake or scorpion bites or goiter, roots and leaves for infertility and herbs for wound healing as well as food while fresh (Sezik et al., 1997; Yesilada & Sezik, 2003). Eryngium species, particularly E. campestre and E. maritimum are called in Turkish folk-medicine as "Bogadikeni" and widely distrubuted in all parts of Turkey. Infusion of aerial parts and roots of these species are used in folk remedies as antitussive, diuretic, appetizer, stimulant and aphrodisiac (Baytop, 1999). Aqueous extract ofthe roots of E. campestre is used orally and poultice is applied externally as a remedy aganst scorpion bite in the Balikesir-Turkey (personal interview). Similar utilizations were found in the old documents from the medieval ages such as against wound and burns, pains, hemorrhoids and sexual diseases (Lev, 2002). Effects of the roots from different Eryngium species against various inflammatory conditions are also well-known in the traditional medicines worldwide; for example, E. campestre as anti-oedema (Leporatti & Ivancheva, 2003) or E. creticum for inflammatory wounds (AliShtayeh et al., 1998). It is also documented in the popular phytotherapy books for such treatments, i.e. E. campestre is recommended against kidney and urinary tract inflammations and oedema (Gruenwald et al. 2000a). To

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confirm the traditional usage ethanolic and aqueous extracts obtained from either aerial parts or roots of E. campestre, E. creticum, E. davisii, E. falcatum, E. isairicum, E. kotschyi, E. maritimum, and E. trisectum were evaluated for their in vivo anti-inflammatory and antinociceptive activities (Kiipeli et al., 2006a). According to the results of investigations, except E. falcatum extracts, ethanol extracts either from the aerial parts or roots of Eryngium species showed apparent anti-inflammatory activity on carrageenan-induced hind paw oedema and TPA-induced ear oedema tests and antinociceptive activity on p-benzoquinone-induced writhing test. In spite of potent activity of the ethanol extract from E. isairicum aerial parts was induced gastric damage. Aerial parts and roots of E. maritimum and E. kotschyii were found to possess most promising activities without including any apparent gastric damage. Moreover, Yesilada et al. (1989) reported that the methanol extract (150 mg/kg) from the whole plant of E. billardieri showed a 44% inhibition on increased vascular permeability induced by acetic acid in mice on oral administration. Further studies were conducted on the various solvent fractions obtained from the aerial parts and roots of the plants and they were tested for their effects on increased vascular permeability induced by acetic acid model, as well as serotonin, bradykinin, and carrageenan-induced paw oedemas in mice. A significant antiinflammatory activity was observed only in the precipitated part of the nbutanol extract of the roots, which mainly contains saponins (Yesilada et al., 1989).

Seseli is an old Greek name that was called by Hippocrates for certain members of the Apiaceae family (Hamlyn, 1969). The genus Seseli L., is composed of aromatic herbs and economically important species that are used as foods, spices, condiments and ornamentals (Lawrence, 1969; Crowden et al., 1969; Pimenov & Leonov, 1993). Several Seseli species are reported in ancient literature for various healing effects. The roots of S. mairei Wolff., have been used against inflammations, swelling, rheumatism, pain and common cold in Chinese traditional medicine (Hu et al., 1990). The seeds of an Indian species, S. indicum, have been reported to possess anthelmintic, carminative, stomachic and stimulant properties (Tandan et al., 1990). In Turkish folk medicine, the fruit of S. tortuosum is used as emmenagogue and anti-flatulence (Baytop, 1999), while the leaves of S. libanotis (Kelemkesir or Kelemenkesir in Turkish) are consumed as a vegetable in the eastern Turkey (Baytop, 1999). Traditional usage of the genus in folk medicine led us to search other species for their biological activies. The ethyl acetate and methanol (80%) extracts obtained from ten Seseli L. species (Apiaceae) growing in Turkey, Seseli andronakii Woron., S. campestre Besser, S. gummiferum Pall. ex Sm. subsp. corymbosum (Boiss & Heldr) P. H. Davis, S. gummiferum Pall. ex Sm. subsp. gummiferum, S. hartvigii Parolly & Nordt, S. libanotis (L.) W. Koch, S. petraeum M. Bieb., S. peucedanoides (Bieb.) Koso-Pol., S. resinosum Freyn & Sint., S. tortuosum

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L. were evaluated for their in vivo anti-inflammatory and antinociceptive activities (Kiipeli et al., 2006c). Among the plant extracts, the ethyl acetate extracts from S. andronakii, S. campestre, S. gummiferum subsp. corymbosum, S. petraeum, S. resinosum and S. tortuosum showed 30.1%, 32.3%, 36.9%, 39.8%, 35.1%, 37.6% inhibition in p-benzoquinone-induced abdominal constriction test, respectively. The ethyl acetate extracts of S. gummiferum subsp. corymbosum, S. petraeum, S. resinosum and S. tortuosum also exhibited notable inhibition, ranging between 24.5-29.7%, 28.1-33.3%, 17.4-27.5%, 27.9-31.3%, respectively, in carrageenan-induced hind paw edema model at 100 mg/kg dose without inducing any gastric damage. During the acute toxicity evaluation, neither death nor gastric bleeding was observed for any of the plant extracts. Results have supported the traditional use of some Seseli species against inflammatory disorders. APOCYNACEAE

Nerium oleander L., oleander, is widely distributed in the Mediterranean region. Due to the toxic nature, limited number of utilization have been recorded in Turkish folk medicine. Among them a speculative assertion on cancer treatment by a Turkish medical doctor became very popular and eventuated recently the leaf extract undergoing Phase I clinical evaluation as a potential treatment against cancer in USA (Smith et al., 2001). During our expeditions, an informant described that fresh oleander flowers was put in alcohol in summer and kept as a home-remedy to alleviate her severe pain and paralysis in her legs. In a reference survey in the database program of Turkish folk medicine (TUHIB), a similar utilization was also encountered by another informant, i.e. flowers kept 40 days in olive oil was applied on joint against rheumatic pain (Yesilada, 2002). Moreover, leaves or flowers are used to stop pain or eczema, sap obtained from fresh leaves for abscess or rheumatism were recorded. Besides, another part of oleander, the fruits, is reported as antirheumatic and as a remedy for skin diseases in Saudi folk medicine (Adam et al., 2001). In this context, Zia et al. (1995) reported that two purified fractions (B 1 and B3 ) obtained from the methanol extract of fresh oleander leaves possess a eNS depressant activity, i.e. produced reduction in locomotor activity, rotarod performance and potentiate hexobarbital induced sleeping time. Moreover, they showed significant analgesic activity as indicated by inhibitory effects on acetic acidinduced writhings and increased reaction time to thermal test. Our results also supported that both aqueous and ethanol extracts of oleander leaves possess significant antinociceptive activity as decided by using another writhing test model, but the effect of EtOH extract was more pronounced (Erdemoglu et al., 2003). However, both extracts were shown gastric ulcerogenicity in mice. On the other hand, EtOH extracts from either dried or fresh flowers exhibited potent antinociceptive activity without inducing any gastric damage and potency was more pronounced than that of reference

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drug aspirin. Experimental results have clearly confirmed that liposoluble fractions of flowers, as decided by ethanol or olive oil extracts were used in folk medicine, possess remarkable antinociceptive activity. Antiinflammatory activity assessment experiment has also verified the conclusion that liposoluble components of flowers may have the activity since only the EtOH extracts were found active even than that of reference drug indomethacin.

ASTERACEAE The genus Achillea, named after the mythological Greek warrior Achilles, comprises of approximately 85 species, most of which are endemic to Europe and the Middle East. In the Turkish flora 40 Achillea species have been recorded and 20 of them are endemic (Davis, 1982). On the otherhand, seveal Achillea species have been known to be used in folk remedies for various purposes such as haemorrhoid and wound healing (Baytop, 1999). Especially, A. millefolium is frequently used against diarrhoea, abdominal pain and stomachache in Turkish traditional medicine (Yesilada et al., 1993; Fujita et al., 1995; Honda et al., 1996). In order to evaluate their folkloric utilization, both antinociceptive and anti-inflammatory activities of five Achillea species including Achillea wilhelmsii C. Koch,A. setacea Waldst & Kit, A. vermicularis Trin., A. phrygia Boiss. & Bal. and A. sipikorensis Hausskn. et Bornm. were investigated (Kiipeli et al., 2007a). The ethanol extracts of A. wilhelmsii, A. setacea and A. vermicularis showed significant antinociceptive (p-benzoquinone-induced writhing test) and antiinflammatory (carrageenan-induced hind paw edema model) activity at 500 mg/kg dose, per os, without inducing any apparent acute toxicity as well as gastric damage. A. phrygia was shown to possess only significant antinociceptive activity, while A. sipikorensis did not show any remarkable anti-inflammatory and antinociceptive activity. Furthermore, the essential oils obtained by water distillation from aerial parts of Achillea schischkinii Sosn. and Achillea aleppica DC. subsp. aleppica were analysed by GC and GC/MS. 1,8-Cineole (32.5% and 26.1%, respectively) was the main component in both oils. Their antimicrobial, anti-inflammatory and antinociceptive activities were also studied and the oil of A. aleppica subsp. aleppica showed significant anti-inflammatory, antinociceptive and moderate antimicrobial activities. The oil of Achillea aleppica subsp. aleppica contains 6.6% of a-bisabolol and its derivatives. These compounds might be at least partially responsible for antiinflammatory activity of the oil (Iscan et al., 2006). In folk medicine, Helichrysum species have been used for gall bladder disorders as medicinal tea, because of their bile regulatory and diuretic effects (Siizge~ et al., 2005). Some of them are used for anti-inflammatory and anti-allergic properties (Carini et al., 2001; Tepe et al., 2005). Moreover,

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this genus is traditionally used in the treatment of wounds, infections and respiratory conditions (Lourens et al., 2004). In Turkey, several Helichrysum species are used in folk medicine as diuretic, cholagogue, and against kidney stones (Baytop, 1999). In order to evaluate the anti-inflammatory and antinociceptive activities of Helichrysum species (Kiipeli et al., 2006b), aqueous extracts from H. arenarium (L.) Moench. subsp. aucheri (Boiss.) Davis & Kupicha and H. plica tum DC. subsp. plicatum were investigated and they showed 37.0%, 30.4% inhibition at 100 mg/kg doses and 31.2%, 28.3% at 200 mg/kg doses in p-benzoquinone-induced abdominal constriction test, respectively, without inducing any gastric damage. The aqueous extracts of all the plants did not show any anti-inflammatory activity in carrageenaninduced hind paw edema model at 50, 100 and 200 mg/kg doses. During the acute toxicity evaluation, neither death nor gastric bleeding was observed for any of the plant extracts.

BERBERIDACEAE Roots and barks of various Berberis species are used as folk remedy for the treatment of various inflammatory diseases such as lumbago, rheumatism and to reduce fever. In Semerkand Bazaar (Uzbekistan) a black tough material prescribed for the treatment of lumbago attracted our attention during our scientific expeditions. The herb dealer described that it was the concentrated aqueous extract of Berberis obionga roots. In a reference survey, several other Berberis species were also found to be used in the treatment of various inflammatory conditions, including rheumatism, fever and pyrexia. In order to evaluate this information, the effect of a widespread Turkish species, B. crataegina DC. was studied and found that the roots of the plant possessed significant anti-inflammatory, analgesic and febrifuge effects. Through bioassay-guided fractionation and isolation procedures berberine was isolated as the main active ingredient responsible from the relevant effects. In that paper, however, it was addressed that the role of other alkaloids in the effect of the roots should also be investigated (Yesilada & Kiipeli, 2002). In the study of Kosar et al. (1999) three types ofisoquinoline alkaloids were detected in the roots, barks and branches of Turkish Berberis species: protoberberine (berberine, palmatine,jatrorrhizine, columbamine), bisbenzylisoquinoline (berbamine, oxyacanthine, aromoline) and aporphine (magnoflorine) types. In a following study, the effects of these alkaloids were studied using various in vivo models in mice (Kiipeli et al., 2002). All alkaloids inhibited inflammations in varying degrees, among them berberine, berbamine and palmatine were shown to possess significant and dosedependent inhibitory activity against serotonin-induced hind paw oedema both on oral and topical applications and acetic acid-induced increase in vascular permeability on oral administration. Moreover, these three alkaloids were also shown to possess dose-dependent antinociceptive activity,

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which assessed by using the model based on the inhibition of p-benzoquinoneinduced writhing movements as well as antipyretic activity on FCA-induced increased rectal temperature on subacute administration. However, all alkaloids induced gastric lesions in varying degrees (Kiipeli et al., 2002). Since it is well known that anti-inflammatory agents acting through inhibition of cyclooxygenase-l (COX-I) enzyme induce such gastric ulceration, the effect of isoquinoline alkaloids might also be based on this mechanism. As a matter of fact, berberine was found to inhibit castor oilinduced diarrhoea in rats, which supported this conclusion (Yesilada & Kiipeli, 2002). Fukuda et al. (1999) demonstrated that berberine inhibits cyclooxygenase-2 (COX-2) transcriptional activity in colon cancer cells in a dose-dependent manner, however, the effect on constitutive enzyme (COX1) was not determined. It is noteworthy that powdered roots of B. lycium is suggested to be taken with milk for the treatment of rheumatism and muscular pains in Pakistan folk medicine, probably to protect the gastric mucosa from damage (Ikram et al., 1966). The results of the previous study (Kiipeli et al., 2002) revealed that berberine, berbamine and palmatine were the main anti-inflammatory, antinociceptive and antipyretic alkaloids in the roots of Berberis species. Especially the effect of first two was more pronounced. Since alkaloid composition ofthe roots showed great variations from species to species, the relevant activity would highly be species-specific. Thus, utilization of B. integerrima roots in therapy would not be beneficial due to the higher concentrations of the alkaloids with weak activity, i.e. magnoflorine and oxyacanthine. On the other hand, due to the higher concentration of effective alkaloids in B. cretica and B. vulgaris roots, more beneficial effects would be expected. Berberine and related isoquinoline alkaloids have been reported to exhibit a broad distribution in the plant kingdom (Keys, 1985), such as; Epimedium sp. leaves (Berberidaceae); Phellodendron sp. barks and Xanthophyllum sp. roots (Rutaceae); Coptis sp. rhizomes and Hydrastis sp. roots (Ranunculaceae); Sanguinaria sp. rhizomes (Papaveraceae); etc. In traditional medicine, some of these plants are known to be employed in the treatment of various inflammatory ailments and results of the present study would also be helpful to explain the relevant effects as well as safety evaluation of these plant remedies especially regarding to the ulcerogenic effect of these alkaloids reported in that study (Kiipeli et al., 2002). Contrary to the observations of the present study that berberine type alkaloids induce remarkable gastric lesions, berberine was reported to be effective in the treatment of gastritis and gastric ulcers in some documents (PDR, 2000), which needs further studies and explanations.

BRASSICACEAE In Turkish folk medicine, Isatis sp. is known to be used for wound healing and against constipation (Baytop, 1999). In Iran, another species of the

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plant, 1. cappadocia, has also been reported against rheumatism, asthma, eczema, fever, headache and wound healing. Rezaeipoor et al. (2000) showed that the plant possess in vivo suppressive effect on humoral primary immune response at the dose of 250 mg/kg, while stimulated secondary immune responses at the dose of 500 mg/kg. Additionally, the activity of a Turkish species, Isatis glauca Aucherx Boiss. ssp. glauca, was studied and ethanol extract demonstrated a significant antinociceptive activity (37.2%) at 500 mg/kg, and a medium anti-inflammatory activity (24-28% inhibition) (Kupeli et al., 2007c).

CISTACEAE In Turkish folk medicine, the leaves of the Cistus laurifolius are also used externally as an effective remedy against several inflammatory ailments such as rheumatic pain, high fever and urinary inflammations. For relieving rheumatic pain a warm decoction of leaves is used as a bath or wilted leaves are externally applied onjoints. For alleviation or treating of urinary inflammations, a poultice is prepared by boiling the leaves and then mixing with flour. This poultice is then applied externally as a plaster on the dorsal part ofthe body in a line with the kidneys (Sezik et al., 1991; Yesilada et al., 1995). Effects of the extracts and fractions from the leaves with non-woody branches ofCistus laurifolius L. (Cistaceae) were studied using two in vivo models of inflammation in mice. For the preliminary antiinflammatory activity assessment of C. laurifolius leaves two types of extracts (aqueous and ethanolic) were prepared and investigated against carrageenan-induced hind paw oedema and acetic acid-induced increased vascular permeability models in mice. Aqueous extract did not show any remarkable effect against carrageenan-induced hind paw edema model, while EtOH extract was significantly active both in 250 and 500 mg/kg doses. A comparable result was obtained against another inflammatory model based on the inhibition of increased vascular permeability, induced by the intraperitoneally injection of acetic acid. Subsequently, through bioassay-guided fractionation and isolation procedures three flavonoids; 3-0-methylquercetin, 3,7 -O-dimethylquercetin and 3,7-0dimethylkaempferol were isolated as the main active ingredients from the ethanol extract (Fig 1). Moreover, these flavonoids were shown to possess potent antinociceptive activity, which was assessed through inhibition ofp-benzoquinone-induced writhing reflex. Results ofthe present study have clearly supported the utilization of C. laurifolius in Turkish traditional medicine and three flavonoids were shown to have strong antinociceptive and anti-inflammatory activities, per os without inducing any apparent acute toxicity as well as gastric damage (Kiipeli & Yesilada, 2007).

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OH Me O

OH

OH

OH

OH

o

0

OH MeO

OH

0

Fig 1. Chemical structures of active compounds

DIOSCOREACEAE Tamus communis root is used to treat rheumatism after pounded in Turkish folk medicine (Yesilada et al., 1995). In a previous report, the ethanol extract from Tamus communis roots was shown to possess significant inhibitory effect on granuloma formation when tested on cotton pellet granuloma model in rats (Capasso et al., 1983; Mascolo et al., 1987). However, in our study neither aqueous nor ethanol extracts prepared from both roots and aerial parts of that plant did show any remarkable anti-inflammatory or antinociceptive activity (Kupeli et al., 2007 c).

ERICACEAE The sap obtained from a fresh branch of Rhododendron ponticum L. is dropped into the toothcave against toothache in Turkish folk medicine (Yesilada et al., 1999). The flowers and fruits of another Rhododendron species, R. molle, have also been recorded in ancient and modern monographs as analgesic and insecticide in Chinese Traditional Medicine (Li et al., 2000). Moreover, the leaves of R. chrysanthum in Siberia (Grieve, 1994) and R. ferrugineum in Germany (Chosson et al., 1998) have been reported as a remedy against rheumatism. In a reference survey, we did not find any experimental study related to this subject, however, Manjeet and Ghosh (1999) reported that quercetin isolated as a major component of R. cinnabarium inhibited LPS-induced nitric oxide and tumor necrosis factor-production in murine macrophages, which might be related with the aforementioned effects. In vivo studies also confirmed that leaves of the

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plant possess a significant antinociceptive activity. The EtOH extract showed the highest antinociceptive activity among the plants investigated as well as a potent anti-inflammatory activity without inducing any gastric lesion was recorded (Erdemoglu et al., 2003). The genus Erica is represented only four species of which one taxon (E. bocquetti (Pe~men) P. F. Stevens) is endemic in Turkey. Among the other species, E. arborea L. and E. manipuliflora Salisb. are widespread species common in the coastal sides in Turkey, while E. sicula Guss. subsp. libanotica (C. & W. Barbey) P. F. Stevens has a limited distribution in South West Anatolia (Stevens, 1978). Herbal tea prepared from aerials parts of E. arborea or E. manipuliflora has been popularly used as diuretic, astringent and treatment of urinary infections in Turkey (Baytop, 1999; Tuzlaci & Erya~ar Aymaz, 2001). In particular, a glass of 5% infusion or decoction of E. arborea leaves is suggested after meals to discharge the edema from the body in slimming formulations (Ba~er et al., 1986; Antonone et al., 1988). In the light of traditional usage anti-inflammatory and antinociceptive activities of different extracts were prepared with methanol, chloroform, ethyl acetate, n-butanol and water from the aerial parts of E. arborea L., E. manipuliflora Salisb., E. bocquetii (Pesmen) P. F. Stevens and E. sicula Guss. subsp. libanotica (C. & W. Barbey) P. F. Stevens (Ericaceae) of Turkish origin were investigated by using in vivo methods (Kiipeli Akkol et al., 2008c). The ethyl acetate extracts of E. arborea (EAE), E. bocquetii (EBE) andE. manipuliflora (EME) exhibited notable inhibition against carrageenan-induced (24.1-32.3%, 23.8-36.1%, 29.2-35.1%, respectively) and PGE 2 -induced (21.2-37.7%, 6.8-29.7%, and 6.2-34.1%, respectively) hind paw edema as well as TPA-induced mouse ear edema models in mice, while the ethyl acetate extract of E. sicula subsp. libanotica (ESE) (10.7-29.7%) displayed potent anti-inflammatory activity only on the PGE 2-induced hind paw edema model. However, the remaining extracts were found to be inactive against inflammatory models. Same extracts i.e., EAE, EBE and EME were also found to exhibit remarkable antinociceptive activity inp-benzoquinone-induced abdominal constriction test at a dose of 100 mg/kg (46.5%,27.7% and 36.3%, respectively) (Kiipeli Akkol et al., 2008c). The anti-inflammatory and antinociceptive activities were mainly determined in the ethyl acetate extracts. Previous studies have shown that flavonoids, anthocyanoids, coumarins, terpenic compounds especially ursolic acid and essential oils were present in Erica species (Vieitez & Ballester, 1972; Ballester et al., 1975; Bennini et al., 1993; Chulia et al., 1995; Dufor et al., 2007). Flavonoids exert significant scavenging properties on oxygen radicals and can interfere with the production of arachidonic acid metabolites trough lipoxygenase enzyme inhibition and reduce the concentration of leukotrienes (Reyes Ruiz et al., 1996; Halliwell & Chirica 1993).

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Calluna vulgaris L. (heather), which is also known as "the true heather of Europe", is the sole member ofthe genus Calluna (Ericaceae) in Turkey (Baytop, 1999). Heather has a long history of medicinal use in folk medicine. In Anatolia, infusion ofthe plant has been used as urinary disinfectant and diuretic (Baytop, 1999). Different ethnobotanical uses ofthe plant have been documented as antiseptic, astringent, cholagogue, depurative, diaphoretic, diuretic, expectorant, mildly sedative, vasoconstrictor and antirheumatic as well as for the treatment of gout symptoms in European societies (Mabey, 1979; Chiej, 1984; Kumarasamy et al., 2002). A homeopathic remedy is prepared from the fresh branches is prescribed in the treatment of rheumatism, arthritis and insomnia (Launert, 1981). The plant is also suggested among the Bach flower remedies and the keywords for prescribing it are "self-centeredness" and "self-concern" (Chancellor, 1985). In order to evaluate this ethnobotanical information, its anti-inflammatory and antinociceptive activities were studied using in vivo experimental models in mice (Orhan et al., 2007b). The ethanolic extract of the plant was first fractionated into five extracts; namely, n-hexane, chloroform, ethyl acetate (EtOAc), n-butanol, and water fractions. Among them, the EtOAc fraction was found to be the most effective and was further subjected to bioassayguided fractionation and isolation procedures. After successive column chromatography applications, on Sephadex LH-20 and silica gel, a component, which is responsible for the above-mentioned activities ofthis species of Turkish origin, was isolated and its structure was elucidated as kaempferol-3-0-[3-D-galactoside, a common flavonol derivative by means of spectral techniques (Fig 2) (Orhan et al., 2007b). A reference survey has revealed that in vitro anti-inflammatory activity of C. vulgaris was previously OH HO

o OH

o

;::O~~

'~ HO

Fig 2. Chemical structure ofkaempferol-3-0-j3-D-galactoside

reported by two separate studies. In the first study, the water extracts of 52 plants from 28 different families, where selections were based upon the records for their use in inflammatory diseases and treatment of wounds in Swedish traditional medicine, were investigated for their inhibitory activities on prostaglandin biosynthesis and platelet activating factor (PAF)-induced exocytosis in vitro and was found highly efficient on both assays (Tunon et

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al., 1995). In particular, C. vulgaris was one of the three plants, which exhibited the most potent cyclooxygenase inhibition in that study. In connection to these data, the flowers of C. vulgaris, was tested in vitro for its effect on arachidonic acid metabolism using mouse peritoneal macrophages, human platelets, and differentiated HL60 leukemic cells (N ajid et al., 1992). Among the several organic and aqueous extracts of heather flowers, the acetone extract was found to inhibit lipoxygenase (LOX) enzyme and further analysis of this extract led to the isolation of ursolic acid as an active anti-lipoxygenase component. Ursolic acid was shown a higher efficiency on 5-LOX inhibition than 12- and 15-LOX, whereas it had a 38% inhibition on cyclooxygenase. Many flavonoids, isolated from medicinal herbs used in traditional medicines, are endowed with desired biological activities su~h as antimicrobial, anti-hepatotoxic, anti-inflammatory, cardioprotective, antiosteoporotic, and anti-cancer (Middleton, 1998). Amongst them, quercetin and kaempferol are known to be the most common flavonols present in many plants in different glycosidic forms. In one study, two coumaryl glycosides ofkaempferol (platanoside and tiliroside) were reported to exhibit strong anti proliferative effect suppressing DNA synthesis in a dose- and time-dependant manner (Dimas et al., 2000). In a very good accordance with the above data (Orhan et al., 2007b), Gil et al. (1994) also figured out that kaempferol-3-0-f3-D-galactoside possessed a remarkable antiinflammatory activity on both in vivo carrageenan-induced hind paw edema and 12-0-tetradecanoylphorbol-3-acetate-induced ear edema as well as in vitro inhibitory effect on phospholipase-~ enzyme. In some similar studies, various kaempferol glycosides have also shown to possess significant inhibitory activities on TNF -{X, production and nitric oxide (NOS) release (Liang et al., 1999; Authore et al., 2001; Matsuda et al., 2001).

GENTIANACEAE Isoorientin is a common C-glycosyl flavone, luteolin-6-C glucoside, and has been reported in many different plant species such as Gentiana, Asphodelus, Rumex, Swertia and Vitex species (Harborne, 1994). During the studies on a Turkish folk medicine, Gentiana olivieri Griseb. (Gentianaceae), isoorientin was isolated as the active anti-hepatotoxic component from the flowering herbs through in vivo bioassay-guided fractionation procedures against carbon tetrachloride-induced hepatic damage (Deliorman-Orhan et al., 2003). In a following bioassay-guided activity assessment study on the same plant material same compound was again isolated as the active hypoglycaemic component (Sezik et al., 2004). In a reference survey, several other biological activities were attributed to isoorientin including potent antioxidant (Ko et al., 1998), antimicrobial (Afifi et al., 1997), myolitic activity on smooth musclecontaining preparations from the rat and guinea pig uterus (Afifi et al., 1999), etc. Isoorientin (Fig 3) was also shown to possess significant

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antinociceptive and anti-inflammatory activities without inducing any apparent acute toxicity as well as gastric damage. Isoorientin induced inhibition between 28.4-37.1 % and 36.4-43.4% against carrageenan-induced inflammation at 15 and 30 mg/kg doses, respectively. The most remarkable point was the anti-inflammatory activity of isoorientin was found almost equal to that of indomethacin (10 mg/kg) which exerted 30.4-40.3% activity, in a very close dose level. Maybe more important point was isoorientin did not induce any visible gastric damage. Due to the high gastric lesion risk of nonsteroidal anti-inflammatory agents, i.e. indomethacin and acetyl salicylic acid, this is a very important peculiarity. On the other hand, isoorientin was also found to possess significant antinociceptive activity, though not potent as acetyl salicylic acid (Kiipeli et al., 2004). Literature survey demonstrated that isoorientin possesses a wide range of biological activities. Due to the adjacent hydroxyl groups at ring B, i.e. catechol functions, the most prominent biological activity reported for isoorientin is a potent antioxidant activity (Diena et al., 2003). Isoorientin showed a strong antiperoxidative activity towards linoleic acid (Mun'im et al., 2003). The antioxidant activity ofisoorientin was also reported by Budzianowski et al. (1991) through evaluating the inhibitory effect on MDA concentration stimulated by Fe+3 ions and ascorbic acid in rat liver microsomes. Ko et al. (1998) studied the antioxidant activity ofisoorientin-6"-0-glucoside, a watersoluble form, from Gentiana arisanensis (Gentianaceae) using various models and reported a potent antioxidant and radical scavenging activity and suggested as an antioxidant therapy against a wide range of free radicalinduced disorders particularly to protect human LDL from oxidative attack. Isoorientin was isolated as one of the active anxiolytic ingredients from a Peruvian folk remedy, Jatropha cilliata M. Arg. (Euphorbiaceae) at 40 and 100 mg/kg doses and the activity at the later dose was identical to that of diazepam. Under the same conditions, a similar effect was observed for orientin obtained from another fraction, however luteoline, aglycone of these two flavonoids, was ineffective. In the same work, analgesic effect was also investigated and isoorientin was found to have more potent effect than orientin (Okuyama et al., 1996). H

OH

H OH

H

H

OH

Fig 3. Structure ofisoorientin

H

H

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GERANIACEAE Geranium species are used as antiasthmatic, antiallergic, antioxidant, antidiarrhoeic, antihepatotoxic, diuretic, tonic, haemostatic, stomachic and antidiabetic in folk medicine (Baytop, 1999). G. pratense ssp. finitimum is particularly used to treat stomachache in in Turkey (Ozaydin et al., 2006). As a relevant activity, G. maculatum has been reported to treat duodenal ulcers as well as in diarrhoea and hemorrhoids in Canada (British Herbal Pharmacopeia, 1987). The leaves of G. thunbergii have been used as an antidiarrhoeic in Japan (Fujiki et al., 2003) and G. sanguineum was reported to possess a potent effect against influenza virus (Sokmen et al., 2005). To obtain an experimental evidence on the therapeutic efficacy of Geranium species and its phenolic compounds for inflammatory diseases, the aqueous extract of aerial parts of G. pratense subsp. finitimum (Woronow) Knuth, its fractions and isolated compounds, i.e. the mixture of quercetin 3-0-aR,

OH

HO

OH

1

R}

R2

OH

H H H H H H H OH

H

2

OH OH

3 5

H

6

OH OH OH

0

R3 a-arabinopyranose f3-galactopyranose f3-glucopyranose f3-galactopyranose f3-glucopyranose (2"-O-galloyl)-f3-glucopyranose (2"-O-galloyl)-f3-galactopyranose (2"-O-galloyl)-f3-glucopyranose OH

~

CI

#

OH

'/11111

0 0H

OH

4 (-)-6-chloroepicatechin

Fig 4. The chemical structures ofthe isolated compounds from Geranium pratense subsp. finitimum

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arabinopyranoside, kaempferoI3-0-[3-galactopyranoside (1), the mixture of quercetin 3-0-j3-glucopyranoside, quercetin 3-0-j3-galactopyranoside (2), kaempferoI3-0-j3-glucopyranoside (3), (-)-6-chloroepicatechin (4), the mixture of quercetin 3-0-(2-0-galloyl)-j3-glucopyranoside, quercetin 3-0-(2-0-galloyl)[3-galactopyranoside (5) and myricetin 3-0-(2-0-galloyl)-j3-glucopyranoside (6) (Fig 4) were examined on carrageenan-, PGE 2 - and TPA-induced inflammation in mice and p-benzoquinone induced writhing reflex to assess anti-inflammatory and antinociceptive activities. The effective dose of materials for the inhibition of carragenan-induced hind paw edema assay was determined to be 100 mg/kg, which was also used in the assays with the extract, its fractions and isolated compounds in all other experiments. The aqueous extract as well as compounds 1 and 2 (100 mg/kg) significantly inhibited the formation of the carrageenan-induced hind paw edema. Significant reductions were also exerted by the aqueous extract and the compounds 1,2 and 5 against PGE 2-induced hind paw edema and TPAinduced ear edema models. The same compounds were shown to possess antinociceptive activity (Kupeli et al., 2007 d). JUGLANDACEAE

Juglans regia L. leaves have been used mostly in worldwide traditional medicines as antifungal, antihelmintic, astringent, keratolytic, antidiarrhoeal, hypoglycaemic, depurative, tonic and for the treatment of sinusitis, cold and stomachache (Girzu et al., 1998; Mouhajir et al., 2001). In Turkish folk medicine, however, leaves are frequently used to reduce fever in sunstroke or to alleviate rheumatic pain, externally. Fresh leaves are spread to form a bed for unclothed patient, who is covered with fresh leaves or pounded with/without raw salt and applied on the naked body or forehead to reduce fever or on swelled joint to alleviate the rheumatic pain, or boiled in water and used for bathing in both conditions (Fujita et al., 1995; Ye~ilada, 2002). Both aqueous and EtOH extracts showed significant antinociceptive activity without inducing any gastric damage, but the effect of later extract was more pronounced than the former as well as that of aspirin (Erdemoglu et al., 2003). EtOH extract also showed a potent antiinflammatory activity as potent as indomethacin. Since direct application of fresh leaf is suggested for the treatment, less polar components of the leaves might possibly be more active (Erdemoglu et al., 2003). LAMIACEAE Prunella vulgaris L. is used against rheumatism, colds and cardiac disorders in Turkish folk medicine (Yesilada et al., 1993). In a previous study, a herbal combination (SKI 306X) consisting of Clematis mandshurica, Trichosanthes kirilowii and Prunella vulgaris, which was used in Chinese traditional medicine for the treatment of various inflammatory disorders, i.e. lymphadenitis and arthritis, was studied for their in vitro effects as anti-inflammatory, analgesic,

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antiarthritic, blood microcirculation-enhancer as well as inhibition of cartilage degeneration enzyme activities on proteoglycan degradation in cartilage explant culture and collagenase-induced rabbit ostheoarthritis model (Choi et al., 2002). The results revealed that SKI 306X inhibited proteoglycan degradation in a concentration-dependent manner and it also showed protective effect on the knee joint of rabbit from ostheoarthritis-like changes on prophylactic administration. In another study, the aqueous extract of P. vulgaris was shown to possess strong antioxidant potency in inhibiting rat erythrocyte hemolysis and lipid peroxidation in rat kidney and brain homogenates (Liu & Ng, 2000). On the other hand, in a study was performed by our group, in vivo anti-inflammatory or antinociceptive activity of the plant was found to be weak (Kupeli et al., 2007). The genus Lamium L. (Lamiaceae) is characterized by almost 40 annual or perennial herbaceous plants spread throughout Europe, Asia, and Africa (Willis, 1973). Lamium album, L. maculatum, and L. purpureum exhibit antispasmodic, anti-inflammatory, antioxidant and antiproliferative properties and have been extensively used in folk medicines as a remedy for the treatment of several disorders such as trauma, fractures, putrescence, paralysis, leucorrhoea, hypertension, as well as women affiictions, such as menorrhagia, uterine hemorrhage, vaginal or cervical inflammations (Bremness, 1995; Matkowski & Piotrowska, 2006; Paduch et al., 2006; Shuya et al., 2003; Weiss, 1988). In Turkish flora, thirty Lamium species have been recorded (Duman, 2000a; Mill, 1982). Among them, L. album, L. maculatum, and L. purpureum have been reported to be used as tonics and for the treatment of constipation in Anatolia (Baytop, 1999). In western Anatolia (in particular in Manisa) whole plant of L. album and several other Lamium species are used to relieve pain in rheumatism and other arthritic ailments (Ozaydin et al., 2006). In order to assess the traditional usage various extracts were prepared from several Lamium species. The n-butanol extracts of L. garganicum subsp.laevigatum (LGL-BuOH), L. garganicum subsp. pulchrum (LGP-BuOH), and L. purpureum var. purpureum (LPPBuOH) exhibited notable inhibition (16.5-28.9%, 14.5-26.9%, 12.3-21.5%, resp.) in carrageenan-induced hind paw edema model at doses of200 mg/kg without inducing any gastric damage. The LGL-BuOH (7.1-30.4%) and LGPBuOH (5.9-24.1%) extracts also displayed potent anti-inflammatory activity against PGE 2-induced hind paw edema model. However, the remaining extracts were found to be inactive against carrageenan-induced hind paw edema and PGE 2-induced hind paw edema models. On the other hand, all the extracts of the title plants failed to display any anti-inflammatory activity on TPA-induced mouse ear edema model. LGL-BuOH and LGP-BuOH were also found to exhibit remarkable antinociceptive activity inp-benzoquinoneinduced abdominal constriction test at a dose of200 mg/kg (25.0% and 24.3%, respectively). The experimental data demonstrated that L. garganicum subsp. laevigatum and L. garganicum subsp. pulchrum displayed remarkable anti-inflammatory and antinociceptive activities (Kiipeli Akkol et al., 2008b).

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Salvia species, Sage, features prominently in the national pharmacopoeias of many countries throughout the world and several species have been used as a remedy against perspiration and fevers; as a carminative; a spasmolytic; an antisepticlbactericidal; an astringent; as a gargle or mouthwash against the oral inflammations such as tongue or throat; a wound-healing agent; in skin and hair care; and against rheumatism (Kintizios, 2000). S. virgata is known as "yilancik" in Turkey and used to alleviate dermatological problems and wounds. The decoction of S. virgata is used against blood cancers in Western Turkey (Baytop, 1999). S. halophila is an endemic plant of Turkey and traditionally being used as herbal tea. To provide a scientific evidence for traditional uses, the aerial parts of S. halophila and S. virgata were subjected to Soxhlet extraction with different solvents i.e. n-hexane, ethyl acetate, methanol and aqueous methanol (50%). Plants were also extracted with water under reflux. The effects ofthe extracts were studied inp-benzoquinone-induced abdominal constriction test for the assessment of antinociceptive activity and carrageenan-induced hind paw oedema and 12-0-tetradecanoyl-13acetate (TPA)-induced ear oedema models in mice for assessing the antiinflammatory activity. Results showed that methanol extract significantly inhibited carrageenan-induced paw oedema and p-benzoquinone-induced abdominal constrictions at 100 mglkg dose, while it was ineffective against the TPA-induced ear oedema. However, the other extracts did not show any inhibitory antinociceptive and anti-inflammatory activities on these in vivo models. The extracts were then analysed using a HPLC-PDA method. Rosmarinic acid was found as the main constituent in the extracts, while caffeic acid and luteolin derivatives were also detected in small ratio (Kiipeli Akkol et al., 2008a). In a literature survey, Hosseinzadeh et al. (2003) reported that the aqueous extract of Salvia leriifolia seeds showed significant and dose-dependent (1.25-10 g/kg) antinociceptive activity over 7 h, and the activity was inhibited by naloxone pretreatment in hot-plate and tail flick tests. They also observed significant and dose-dependent (2.510 glkg) activity against acute inflammation induced by acetic acid and in the xylene ear oedema test. In the chronic inflammation test (cotton pellet test), however, the extract (2.5-5 g/kg) demonstrated considerably significant and dose-dependent anti-inflammatory activity. Consequently, a supraspinal antinociceptive effect mediated by opioid receptors has been suggested as the activity mechanism of extract (Hosseinzadeh et al., 2003). On the other hand, in a previous study, the methanol extract of Salvia miltiorrhiza was found to inhibit production ofPGD 2 and the ethyl acetate subfraction exerted the strongest inhibition. From the ethyl acetate subfraction, tanshinone I was isolated as an active principle through activity-guided isolation procedures (Kim et al., 2002). The genus Sideritis L., widely distributed in MediterraneanMacronesian region, are traditionally used in Spanish folk medicine for their anti-inflammatory and gastroprotective properties to treat certain

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disorders that are accompained by inflammation. Several anti-inflammatory compounds have been obtained from the members of this genus, mainly flavonoids and terpenoids (Godoy et al., 2000). In Turkey, this genus is represented by 46 species (Duman, 2000b; Ayta!; & Aksoy, 2000) and some of which are used in the treatment of gastrointestinal ailments, common colds (including bronchitis, sore throat, flu) and as a diuretic as well as a herbal tea for pleasure in Turkish folk medicine (Baytop, 1999; Yesilada et al., 1995). In the light oftraditonal usage, acetone extract from the aerial parts of Sideritis ozturkii Aytac & Aksoy and its fractions were investigated for its in vivo anti-inflammatory and antinociceptive activities. Acetone extract of the plant and its phenolic fraction were found to possess significant inhibitory activity on carrageenan-induced hind paw oedema and pbenzoquinone induced writhing reflex models in mice. Ozturkoside A (chrysoeriol 7-0- [2'"-0-caffeoyl-6'"-O-acetyl-13-D-glucopyranosyl-( 1~2)-13- Dglucopyranoside]) (1), ozturkoside B (chrysoeriol 7-0-[2"'-0-caffeoyl-13-Dglucopyranosyl-(1~2)-13-D-glucopyranoside]) (2) and ozturkoside C (chrysoeriol 7-0- [2'"-0-p-coumaroyl-6'"-O-acetyl-13-D-glucopyranosyl-( 1~2)13-D-glucopyranoside]) (3) were isolated from the active phenolic fraction (Fig 5). Ozturkoside C showed notable antinociceptive and anti-inflammatory activities without inducing any apparent acute toxicity or gastric damage.

OH

OH

HO

0

a

Fig 5. Chemical structures of compounds 1-3

Although the activity of ozturkosides A and B were not significant in statistical analysis, some inhibitory effect was observed. Accordingly, it is suggested that these components in phenolic fraction might possibly share the antinociceptive and anti-inflammatory activities together (Kiipeli et al., 2007b). In spite of a high number of studies reporting the anti-inflammatory and antinociceptive activities of several Sideritis species only two flavonoids have been isolated and defined as the active constituents; hypolaetin-8-

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glucoside (Villar et al., 1984) and 5-0-demethylnobiletin (Bas et al., 2006). As shown in Fig 6, hypolaetin-8-glucoside shows very close chemical structure to ozturkoside C, both having luteoline type flavone glycoside structure. OH OH O-Glu HO

o OH

Fig 6. Chemical structure ofhypolaetin-8-glucoside

Additionally, acetone extract of S. stricta Boiss. & Heldr. apud Bentham and its phenolic fraction exhibited potent inhibitory activity against both bioassay models in mice. From the active phenolic fraction a well-known phenylethanoid glycoside, verb as cos ide (= acteoside) (1) and two flavonoid glycosides, isoscutellarein 7-0- [6'" -O-acetyl-~- D-allopyranosyl(1 ~2)l-~-D-glucopyranoside (2), and isoscutellarein 7-0-[6'" -O-acetyl-~-D­ allopyranosyl-( 1~2)l-6f1-0-acetyl-~- D-glucopyranoside (3) were isolated. During phytochemical studies we also isolated a methoxyflavone, xanthomicrol (4) from the non-polar fraction (Fig 7). Although antinociceptive and anti-inflammatory activities of phenolic components were found not significant in statistical analysis, compounds 1 to 3 showed a notable activity without inducing any apparent acute toxicity as well as gastric damage. Furthermore, a mixture of flavonoid glycosides (2 + 3) exhibited a significant inhibitory effect in both models at a higher dose (Kiipeli et al., 2007c).

HO

HO

1

87

Effects of Medicinal Plants in Turkey Fig 7. Contd. R OH HO

OCOCH3

o o

HO OH

2 R:OH 3 R: OCOCH 3 4'

OH

OH

0 4

Fig 7. Chemical structures of compounds 1 - 4

LAURACEAE Laurus nobilis L., laurel, is a spice plant widely distributed in the Mediterranean area and the seed oil is used against rheumatic pain in Turkish folk medicine as well as in Europe. Previously, the oil obtained from laurel fruit was also reported to be used for treatment of furuncles, sprains, bruises and rheumatism (Simic et al., 2003). The experimental results indicated that the ethanol extract of the seeds possesses a remarkable anti-inflammatory (29.1-37.3%) and antinociceptive (34%) activity at a dose of 500 mg/kg (Kupeli et al., 2007c). On the other hand, extract prepared from the leaves were also studied for comparison and was found completely devoid of any activity (Kupeli et al., 2007 c).

LORANTHACEAE The genus Viscum L. (Loranthaceae) comprises semi-parasitic plants which grow on various host tree and shrubs (Miller, 1982). Viscum album L. is the most widespread species worldwide and has been reputed against cardiovascular diseases, i.e. hypertension and atherosclerosis; various bone and joint disorders including periarthritis, spondylitis, and arthritis; to

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alleviate headache; for immune system stimulation; in nervous disorders as sedative or to combat epilepsy (Bartram, 1995; Murray, 1995; Wichtl & Bisset, 1994). In order to evaluate this information, antinociceptive and anti-inflammatory activity ofthe five flavonoids [5,7 -dimethoxy naringenin (1,3, and 5) or 4', 6'-dimethoxy chalcononaringenin (2, 4)] derivatives; (5,7dimethoxy-flavanone-4' -0- ~- D-gi ucopyranoside (1), 2'-hydroxy-4' ,6'dimethoxy-chalcone-4-0-~- D-glucopyranoside (2), 5,7 -dimethoxy-flavanone4'-0- [2"-0-(5111-0-trans-cinnamoyl)-~-D-apiofuranosyl]-~- D-glucopyranoside (3), 2'-hydroxy-4' ,6'-dimethoxy-chalcone-4-0-[2"-O-(5'"-O-trans-cinnamoyl)-~­ D-apiofuranosyl]-~- D-glucopyranoside (4), 5,7 -dimethoxy-flavanone-4'-0- [~­ D-apiofuranosyl(I~2)]-~-D-glucopyranoside (5) isolated from ethyl acetate fraction of V. album ssp. album were investigated (Fig 8). The ethyl acetate fraction showed a remarkable and dose-dependent anti-inflammatory activity on carrageenan-induced hind paw oedema model (between 24.7 to 37.2%). Same pharmacological profile was also observed for the compound 2 (30.7-33.6%) from the ethyl acetate fraction. The anti-inflammatory activity of 2 was almost as potent as that of indomethacin which exerts 33.3-42.7% anti-inflammatory activity. Other flavonoids, 1, 3, and 5, isolated from the ethyl acetate fraction, also showed significant but weaker antiinflammatory activity against the same model. Among the isolated compounds from the ethyl acetate fraction were administered in 30 mg! kg dose, compounds 2 and 5 were found to possess highest and significant antinociceptive activity on p-benzoquinone induced writhing reflex test, but not as potent as ASA. The ethyl acetate fraction and isolated compounds did not induce any apparent acute toxicity during the 24 h observation period. It is noteworthy that, in spite of a weak gastric lesion incidence in the higher dose of ethyl acetate fraction (gastric lesions were observed in the stomach of lout of 6 rats), all the isolated flavonoids were found completely safe from the view point of gastric damage (Deliorman et al., 2006). Herencia et al. (1999), investigated the effects of a series of chalcone derivatives on various in vivo and in vitro inflammatory models (i.e. human neutrophil functions, eicosanoid release, TNF -ex production, air-pouch, etc.) and reported inhibitory effects on iNOS and COX-2. Correa et al. (2001) reported potent antinociceptive activity for 3,4-dichlorochalcones using the writhing test in mice. In the study of Pelzer et al. (1998) it was concluded that the anti-inflammatory activity offlavonoids increase depending upon the catechol or guaiacol-like substitution to Bring (3', 4'-dihydroxy or 3"hydroxy-4'-methoxy or 3'-methoxy-4'-hydroxy). Accordingly flavanones such as eriodictyol, 7-0-methyleridictyol and hesperidin showed the highest activity against carrageenan-induced edema. However, flavanone and chalcone derivatives demonstrated to possess anti-inflammatory activity in the present study were shown to possess one glucosylated hydroxyl group at Bring [5, 7-dimethoxy naringenin (1, 3, and 5) or 4', 6'-dimethoxy chalcononaringenin (2, 4) derivatives]. Therefore, results showed herein present additional structural data for the anti-inflammatory

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and antinociceptive activity evaluation of chalcone and flavanone derivatives.

8

°

~9

6

5

~o

4

° (1)

HO HO~OH

14°"f-°----i-CH20H OH

5

°

(2)

°

°

or~/~f HO HO

(4)

Fig 8. Contd.

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Fig 8. Contd.

HO~'''' ~"

I

1 """"-

2

~'HO OH

3' 4'

6

0

1" 0

CH20H

5

3

(5)

Fig 8. Structures of investigated compounds

Arceuthobium oxycedri (DC.) Bieb., is a semi-parasitic plant grows on Juniperus oxycedri and the shape and color ofthe plant looks very similar to host plant. In Anatolia this plant is believed to be effective against a wide range of diseases; from inflammatory to infectious (Yesilada et al., 1995; Fujita et al., 1995; Honda et al., 1996). To confirm the conventional usage, prepared ethanol and aqueous extracts of the plant were studied and both extracts exhibited significant anti-inflammatory effect at the ranges of 28.9-34.2% and 21.1-30.5%, respectively. The plant also showed a potent antinociceptive activity; for the aqueous extract 34.2% at 500 mgt kg dose, while for the ethanol extract 28.6% at 250 mg/kg and 39.9% at 500 mg/kg doses. Results supported the folkloric utilization of the plant as no such study has been carried out on this plant previously (Kupeli et al., 2007c).

LYCOPODIACEAE Lycopodium genus (syn. Huperzia), commonly known as "club moss, ground pine, devil's claw, devil ash" in English, is a pteridophyte abundantly found in subtropical and tropical forests in the world, which is under the risk of extinction (Lawrence, 1989). In Turkey, the genus is represented by five species, namely L. alpinum L., L. annotinum L., L. clavatum L., L. complanatum ssp. chamaecyparissus (A. Br.) Doll, andL. selago L. (Davis & Cullen, 1984). Among them, L. clavatum (LC), and has been reported to be used for wound-healing effect as well as against nappies occurring in babies and, therefore, also called "belly powder" (Baytop, 1999). The spores ofthe plant were stated to have a protective effect as dusting powder for tender skin (Vasudeva, 1999). In Papua New Guinea, LC is used against stomach pain (Holdsworth & Giheno, 1975). Some other Lycopodium species have also been reported for similar or related ethnobotanical utilizations

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worldwide. According to an ethnobotanical survey conducted in Sabah region of Malaysia, Lycopodium sp. was recorded to be used to treat skin burns (Kulip, 1997). In Wisconsin (USA), the leaf decoction of L. obscurum was reported to be used against muscle pain and rheumatism (Coffey, 1993). Studies on four extracts prepared with petroleum ether, chloroform, ethyl acetate and methanol as well as the alkaloid fraction from the aerial parts of L. clavatum L. using acetic acid-induced increase in capillary permeability assessment in mice revealed that only the chloroform extract and the alkaloid fraction displayed marked anti-inflammatory effect at a dose of 500 mg/kg having percentage of inhibition 24.3 and 32.1, respectively, as compared to indomethacin which exhibited 44.6% of inhibition at 10 mg/kg dose. The alkaloid fraction of L. clavatum was submitted to bioassay-guided fractionation procedures and revealed that the alkaloidal-type of compounds might possibly be responsible for the anti-inflammatory activity, which supports the folk medicinal utilization of the plant. Gas chromatographicmass spectrophotometric analysis of the active alkaloid fraction have demonstrated that lycopodine, an alkaloid isolated from several Lycopodium species, was the major alkaloid component (84.5%) (Fig 9). Accordingly, these experimental results point out that the anti-inflammatory activity of the alkaloid fraction of the aerial parts of Lycopodium clavatum are primarily due to the alkaloidal components, which might most probably be lycopodine as the major responsible compound (Orhan et ai., 2007a).

Fig 9. Chemical structure oflycopodine

LYTHRACEAE

Lythrum saiicaria (purple loosestrife) was known as medicinal plant already in ancient Greek and Roman times and it has been an important drug for centuries. The whole flowering plant and the flowering branch tips of this

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plant are used not only in folk medicine but also in pharmaceuticals. Decoction or some other fluid extracts from Lythrum salicaria is used internally for diarrhoea, chronic intestinal catarrh, hemorrhoid and eczema. It is also used externally to treat varicose veins, bleeding of the gums, hemorrhoids and eczema (Baytop, 1999; Mantle et al., 2000; Rauha et al., 2000). Dried aerial parts of L. salicaria L. were sequentially extracted with different solvents such as petroleum ether, ethyl acetate, methanol and 50 % aqueous methanol. Water extract of L. salicaria was also prepared under reflux. The extracts were then submitted to bioassays, i.e. free radical scavenging activity [1,1-diphenyl-2-picrylhydrazyl, DPPH% assay, iron(IlI) reductive activity, capacity of the inhibition of linoleic acid peroxidation and MDA formation], antinociceptive activity fp-benzoquinone-induced abdominal constriction test] and anti-inflammatory activity [carrageenaninduced hind paw edema model]. In addition, the content oftotal phenolics, flavonoids and flavonols in the extracts were determined by spectrophotometric methods. Although the methanol extract showed antiinflammatory and antinociceptive activity other extracts remained inactive. None of the extracts caused any gastric damage. The qualitative and quantitative compositions of all the extracts were analysed using a HPLCPDA system, polar fractions were found to be rich in flavonoids such as isovitexin and isoorientin (Tunalier et al., 2007). In a literature review, L. salicaria tannins, flavone-C-glycosides and anthocyanins have been identified in L. salicaria extracts. Isoorientin, isovitexin and their derivatives have been reported as its main flavonoids (Rauha et al., 2000; Rauha, 2001). Rauha (2001) and Tunalier et al. (2007) reported the concentration of flavonoids in L. salicaria to be high and isoorientin was reported to possess anti-inflammatory while isovitexin antioxidant activities (Ko et al., 1998; Arimoto et al., 2000; Kiipeli et al., 2004). MORACEAE

Maclura pomifera (Rafin.) Schneider (syn. M. aurantiaca Nutt.), Ioxylon pomiferum Raf., Toxylon pomiferum Raf. ex Sargis a widely cultivated hardwood tree in Turkey for ornamental purposes. Various parts of the Maclura species are used in folkloric medicine worldwide. Decoction prepared from the roots of M. pomifera is used for the treatment of sore eyes by Comanche Indians in the North America (Carlson & Volney, 1940). The sap ofthe plant is used for the treatment of toothache and the barks and leaves for uterine haemorrhage in Bolivia (Bourdy et al., 2004). While the bark of M. tinctoria has been reported to be used against toothache by Kaiowa and Guarani indigenous people living in the Caarapo Reserve in Brazil as well as the in the other parts of Amazon region, it was also recorded to be used

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in southern Ghana for dental health (Elwin-Lewis et al., 1980; Elvin-Lewis & Lewis, 1983; Bueno et al., 2005). Aqueous, ethanolic and chloroform extracts and two prenylated isoflavones; scandenone CD and auriculasin (II), isolated from the fruits of Maclura pomifera, were investigated for their in vivo anti-inflammatory and antinociceptive activities. The ethanol and chloroform extracts and scandenone had 37.2%, 42.0% and 29.7% inhibition, respectively, while acetylsalisilic acid (ASA), the reference compound, showed 53.5% inhibition at 100 mg/kg dose. Extracts and scandenone also exhibited significant inhibition, ranging between 23.3-41. 7% for the ethanol and 16.940.3% for the chloroform extracts as well as 23.9-35.4% for scandenone in carrageenan-induced hind paw edema model at 100 mg/kg dose. All the extracts and compounds were found completely safe from the viewpoint of gastric damage and no acute toxicity was observed within 48 h observational period. A similar activity pattern was also observed in TPA-induced ear edema model, the EtOH extract (49.2% and 54.9%), chloroform extract (45.1 % and 49.6%) and scandenone (45.7% and 49.3%) displayed potent antiinflammatory activity as measurement of edema weight as well as swelling thickness, compared to indomethacin (62.7% and 70.5%). However, the H 20 extract and auriculasine were found to be completely inactive against this model. Results of the present study have revealed that isoflavonoids are the major anti-inflammatory and antinociceptive principles of the fruits. Scandenone showed a potent activity profile against all the models employed, while that of other isoflavonoid, auriculasin, was not significant, but within reasonable limits, i.e. 20.6% on p-benzoquinone-induced writhing test (Kiipeli et al., 2006). Among the various biological activity profiles reported for flavonoids so far, potent antioxidant activity was particularly attributed to the existence of a catechol group in the B ring (Brandi, 1997; Hom-Ross et al., 2003; Tsao et al., 2003; Cioffi et al., 2003). Since inflammation has been found to elevate the levels offree radicals, antioxidant agents might be also considered possibly to display anti-inflammatory effect (Guo et al., 2002). As shown in Fig 10, auriculasin possesses a catechol group in the molecule; however the anti-inflammatory activity was less potent, not significant, than that of scandenone which does not possess this function. Tsao et al. (2003) studied the antioxidant activity ofisoflavones from M. pomifera fruits. Accordingly, they isolated two linear isoflavones; osajin and pomiferin, as the main constituents from the ethyl acetate extract. These compounds were chemically constitutional isomers of scandenone and auriculasin. Pomiferin, possessing catechol group on ring B, was found to display a strong antioxidant activity, as expected, comparable to the antioxidant vitamins C and E, while osajin (bearing single hydroxyl group in ring B), genistein and daidzein showed no antioxidant activity.

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o R

OH

o

R=H (scandenon) R=OH (auriculasin)

OH

Fig 10. Chemical structures of scandenone and auriculasin

POLYGONACEAE Rumex crispus L. is reported to treat headache, wound healing and to promote maturation of abscess in folkloric medicine in Turkey (Yesilada et al., 1995). Besides, a literature survey revealed that several other Rumex species have also been traditionally used in the treatment of eczema, wound healing, swelling, tonsillitis, leprosy and hemorrhoids in Ethiopia or as analgesic and antipyretic in India and to treat burns and to remove pain from the back and lumber region in Bangladesh (Gebrie et al., 2004; Ghosh et al., 2003; Rouf et al., 2003). In a previous study on the anti-inflammatory activity oftwo Rumex species based on the cell viability and measurement ofPGE 2 concentration, the results verified that the root extract of R. abyssinicus to inhibit PGE 2 synthesis, whereas the leaf extract of R. nervosus showed no inhibition at all (Getie et al., 2003). In another study, anti-inflammatory activity of R. patientia, a plant used for wound healing and anti-inflammatory properties in Turkish folk medicine, was studied against carrageenan, histamine, dextrane, serotonin, formaldehyde-induced oedema, cotton pellet granuloma and Kabak test models in rats and the extract was found to possess significant anti-inflammatory activity which was concluded to be dependant on its rich anthraquinone and tannin content (Suleyman et al., 1999). Conversely, the experiments performed by our group, demonstrated that both aqueous and ethanol extracts of R. crispus displayed no remarkable anti-inflammatory or antinociceptive activity (Kupeli et al., 2007). RANUNCULACEAE The Clematis species have been extensively used in the traditional medicines worldwide due to its widespread distribution at the northern hemisphere. The aerial parts of various Clematis species are particularly used in Europe

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and Eastern Asia as a remedy to reduce pain and fever, as diuretic, used in the treatment of rheumatic pain, eye infections, gonorrhoeal symptoms, bone illnesses, chronic skin disorders, gout and varicosity (Keys, 1985; PDR, 2000). Several species of Clematis have been used as remedy in Anatolia as well. The most salient application related to folkloric usage is the one that is applied in the treatment of rheumatic ailments. In Northern Anatolia, C. cirrhosa vs C. flammula leaves or the aerial part are used to provide temporary relief of joint pains. Ground fresh aerial parts or leaves are applied on inflammatory joints for about 15-30 minutes. The ensuing irritation on the skin opens a gap and drains the edema. In some particular cases, the wound is plugged by inserting a grape dregs in order to provide continuous drainage of the edema for 20-25 days. The plug is removed occasionally to drain the accumulated inflammation out. Then, to cure this open wound a fresh leaf of Plantago major ssp. major L. is applied (Yesilada et al., 1991). Moreover, C. vitalba branches are also used to stop tooth pain by smoking like a cigarette in northwestern Anatolia (Yesilada et al., 1999). In order to test the effectiveness of extracts, fractions and subfractions from dried Clematis vitalba L. aerial parts were studied in mice. Ethanolic extract exerted a potent effect on carrageenan-induced hind paw edema and acetic acid-induced increased vascular permeability models. Through bioassay-guided fractionation procedures a new C-glycosyl flavone, 4'-0coumaroyl-isovitexine (vitalboside) was isolated as the main active ingredient ofthe aerial parts (Fig 11). Vitalboside showed a potent and dose-dependent (in 75 and 150 mg/kg does, per os) in vivo anti-inflammatory activity against acute (carrageenan-, serotonin- and PGE 2-induced hind paw edema model, castor oil-induced diarrhoea), subacute (subcutaneous air-pouch) and chronic (Freund's complete adjuvant-induced arthritis) models of inflammation. The same compound was also isolated as the main antinociceptive principle which was assessed by using the models based on the inhibition ofp-benzoquinoneinduced writhings, as well as antipyretic activity against Freund's complete adjuvant-induced increased body temperature. Acute and subchronic toxicity studies were also performed (Yesilada & Kiipeli, 2007). Previously, Li et al. (2002,2003) studied the in vitro activity of several plants on cyclooxygenase1 (COX-l) enzyme, one of the key enzymes in inflammatory pathway, including Clematis species which are used against various inflammatory disorders such as asthma, arthritis, rheumatism, fever or snake bite in Australia and China. They reported that C. chinensis and C. glycinoides stems did not show any apparent activity against COX-1 enzyme, while C. pickeringii leaves were found to possess a potent inhibition. In spite of the worldwide practice of using dried aerial part of Clematis species against inflammatory disorders in traditional medicines, fresh aerial part of Clematis species is known to possess severe irritating in nature. Such irritation caused by the enzymatic transformation of the ranunculin glycoside into protoanemonin after crushing the fresh plant (PDR, 2000). Ranunculin and eventually protoanemonin which are also known as the most characteristic

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components of some other Ranunculaceae plants such as Ranunculus, Anemone or Helleborus species are ultimately irritating and volatile substances (Didry et al., 1993; Southwell & Tucker, 1993). In fact, some Ranunculus species are also practiced in the same way as of Clematis species for the same purpose among locals in Anatolia (Yesilada et al., 1995). Protoanemonin is an utterly weak substance and transforms into anemonin after being rapidly dimerized. On the other hand, no protoanemonin has been found in the dried plant (PDR, 2000). At the monograph of the plant (Traveller's Joy) in PDR Gruenwald et al., (2000) for Herbal Medicines it is reported that no side effect was observed on either internal or external application of the dehydrated plant, while even contact with the crushed or cut fresh aerial part induces irritation on the skin. Accidental consumption ofthe fresh aerial part leads to strong irritation at the gastrointestinal and urinary tract and provokes colic, diarrhoea and related symptoms. Higher doses are reported to induce death on experimental animals depending upon the protoanemonin content in the plant. On the other hand, Pieroni et al. (2002) reported that C. vitalba sprouts are boiled in large amounts of water in order to cleave protoanemonin prior to eaten in central Italy.

OH

OH Fig 11. Chemical structure of 4' -O-coumaroyl-isovitexine <=vitalboside)

In Turkish folk medicine, Helleborus orientalis Lam. roots are used to treat edema in cattle. A small piece of root is sharpened like a nail is inserted into a hole punched in the ear/or tail of cattle and kept 24 h to reduce inflammation in extremities or applied externally on swelled joints or dried and pounded root is orally administered (Fujita et al., 1995; Yesilada, 2002). Alternatively, the extract of roots mixed with Salvia tomentosa herbs is used as a bath for both man and animals. Although roots ofthe plant are preferentially used to alleviate the inflammatory conditions in cattle, it is not restricted to animals. A small piece of root is inserted inside a cavity against toothache in man (Fujita et al., 1995). The utilization of another Helleborus species, H. purpurascens, for the treatment of inflammatory stages of rheumatism and autoimmune diseases has also been reported in Romania (Olinescu et al., 1986). This plant was also shown to have in vivo anti-inflammatory activity in rheumatism as well as immunosuppressant

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activity (Linke et al., 1998). Another species, H. niger, was also reported to possess immunomodulating properties (Bussing & Schweizer, 1998). In order to assess the anti-inflammatory and antinociceptive activities aqueous and ethanolic extracts were prepared. Our experiments proved that both roots and herbs of H. orientalis to possess significant antinociceptive activity, but that of EtOH extract was more pronounced and did not induce any gastric damage. However, only the EtOH extract of roots showed potent antiinflammatory activity (Erdemoglu et al., 2003). Ranunculus species are used in Turkish folk medicine against rheumatic pain, in particular to drain the pus from the inflammed joint through applying the fresh flowering herb after pounding on joint for about 20 min (Baytop, 1999; Yesilada et al., 1995). In an in vivo study, ethanol extract from R. tricophyllus Chaix herb showed a weak anti-inflammatory and antinociceptive activity (Kupeli et al., 2007).

ROSACEAE The leaves of Laurocerasus offficinalis Roemer are used for asthma, coughs, indigestion and dyspepsia (Grieve, 1994). The fresh leaves of this plant is used in Turkish folk medicine as analgesic for headache and as antipyretic externally. Fresh leaves are applied on the forehead, after being wilted over a fire (Yesilada et al., 1999). Conventional usage of this plant in folk medicine led us to search the biological activity of the plant extracts in a scientific platform. Even though the activity of aqueous extract was weak, EtOH extract showed potent antinociceptive and anti-inflammatory activity without inducing any gastric lesion which supports the claimed activity (Erdemoglu et al., 2003). Different parts of Rubus species are used in folk medicine for the treatment of numerous diseases such as diabetes mellitus, inflammatory disorders, diarrhoea, hemorrhoids and ulcers (Jouad et al., 2002; Marquina et al., 2002; Panizzi et al., 2002). Recently, the novel anti-inflammatory triterpenoids, tormentic acid and euscaphic acid, were isolated from the fresh leaves of R. sieboldii (Murakami et al., 2002). In a previous study, the aerial parts of R. sanctus, R. hirtus and their hybrid were evaluated for anti-inflammatory activity using carrageenan-induced hind paw edema in mice and polar fractions, n-butanol and remaining aqueous fractions obtained by solvent extraction, were shown to possess significant activity (Akcos et al., 1998). Decoction ofthe roots Rubus sanctus is used to alleviate pain and against rheumatism as tea (Honda et al., 1996). In an experimental study, EtOH extracts of R. sanctus Schreber (root and aerial part) and R. hirtus Walds. et Kit (aerial parts) showed potent antinociceptive activity, while that of aqueous extracts were weak. However, it should be noticed that both plants extracts may induce gastric damage. Meanwhile, neither roots nor aerial parts of both species showed a significant anti-inflammatory

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activity against carragenan-induced hind paw edema in 500 mg/kg dose (Erdemoglu et al., 2003). In the German Commission E Monographs, fruits (rose-hips, with seeds) of Rosa canina L. (Rosaceae), the dog rose, are stated to possess prophylactic and therapeutic activities against a wide range of ailments, including the inflammatory disorders arthritis, rheumatism, gout, and sciatica, for diseases with fever; for colds and infectious diseases including influenza, against gastro-intestinal disorders, to aid digestion, prevention of inflammation of the gastric mucosa and gastric ulcer, for gallstones, biliary complaints, as a laxative, for disorders of the kidney and the lower urinary tract, as a diuretic, for dropsy and as an astringent (Blumenthal et al., 1998). The plant has a widespread distribution in Turkey and was reported as the most commonly utilized remedy following Plantago species in Turkish folk medicine (Yesilada, 2002). In addition to the effects offruits against the disorders described above, the fruit is known as the most effective remedy against hemorrhoids and diabetes mellitus in Turkish folk medicine. Besides, the roots and leaves of the plant have also been used against bronchitis (Tabata et al., 1994; Fujita et al., 1995; Yesilada et al., 1995, 1999; Honda et al., 1996; Sezik et al., 2001). This knowledge led us to analyze the plant extracts for their biological activity. The aqueous and ethanol extracts of Rosa canina L. (Rosaceae) fruits and the fractions prepared from the latter were investigated for their anti-inflammatory and antinociceptive activities in several in vivo experimental models. The ethanolic extract was shown to possess significant inhibitory activity against inflammatory models (i.e. carrageenan-induced and PGE1-induced hind paw edema models, as well as on acetic acid-induced increase in a capillary permeability model) and on a pain model based on the inhibition of pbenzoquinone-induced writhing in mice. The active EtOH extract was then fractionated through solvent extractions with increasing polarity and five fractions were obtained; hexane, CHC13 , ethylacetate (EtOAc), n-BuOH, and the remaining water fraction. Ethylacetate and n-butanol fractions displayed potent anti-inflammatory and antinociceptive activities at a dose of 919 mg/kg without inducing acute toxicity. Further attempts to isolate and define the active anti-inflammatory and antinociceptive component(s) from the EtOAc and n-BuOH fractions through bioassay-guided isolation procedures have resulted in failure. Both fractions were further submitted to chromatographic separation, but the activities weakened to a large extent, probably due to the synergistic interaction of components in the active fractions (Deliorman Orhan et al., 2007).

SCROPHULARIACEAE Since the ancient times, Verbascum extracts, decoctions and infusions, commonly known as "mullein", have been used as medicinal herbs in traditional medicines worldwide. The leaves and flowers of Verbascum are

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reported to have expectorant, mucolytic and demulcent properties which are used to treat respiratory problems such as bronchitis, dry coughs, tuberculosis and asthma in traditional Turkish medicine (Baytop, 1999; Turker & Camper, 2002). Verbascum flowers are boiled in milk and are applied externally for pruritic conditions affecting the urogenital organs. In addition, as a traditional Anatolian cure for anal fistulae, the flowers are boiled in a cauldron and then the anus is exposed to the vapors. These species are reported to be mildly diuretic and to have a soothing and antiinflammatory effect on the urinary tract, as well as acting as a mild sedative. Oil made from the flowers is used soothing earache, and can be applied externally for eczema and other types of inflammatory skin conditions. These species are commonly used to treat hemorrhoids, rheumatic pain, superfacial fungal infections, wounds and diarrhoea. They are traditionally consumed as a tea to relieve abdominal pains (Tuzlaci & Erol, 1999; Sezik et al., 2001; Turker & Camper, 2002). In addition to the above mentioned common uses for the Verbascum family without referring a particular species, Verbascum lasianthum Boiss. ex Bentham flowers are reported to be used against hemorrhoids in southwest Anatolia (Tuzlaci & Erol, 1999). To evaluate the scientific basis for this practice, in vivo anti-inflammatory and antinociceptive activities were investigated. A methanolic extract of the flowers was shown to possess significant inhibitory activity in the carrageenan-induced hind paw oedema model and in p-benzoquinoneinduced writhings in mice. Through bioassay-guided fractionation and isolation procedures eight compounds, 6-0-(4"'-0-trans-p-coumaroyl}-a- Lrhamnopyranosylaucubin (1), 6-0-(4'" -O-trans-p- methoxycinnamoyl}-a-Lrhamnopyranosylaucubin (2), sinuatol (3), aucubin (4), geniposidic acid (5), catalpol (6), ajugol (7) and ilwensisaponin A (8) were isolated and their structures were elucidated by spectral techniques (Fig 12). An iridoid glucoside, aucubin (4) and a triterpenoid saponin, ilwensisaponin A (8) were found to possess significant antinociceptive and anti-inflammatory activities, per os without inducing any apparent acute toxicity or gastric damage (Kupeli et al., 2007a). Aucubin, one ofthe major iridoid glycosides of the flowers of Verbascum lasianthum, was previously shown to inhibit the TNF-a and IL-6 production in RBL-2H3 mast cells. Aucubin induced an enlarged IkE pool through inhibition ofIkE inhibited translocation ofTNFKBIRel A to nucleus, and therefore inhibited expression of inflammatory cytokines TNF -a and IL-6 (Jeong et al., 2002). Moreover, Recio et al. (1994) reported that aucubin was effective against carrageenan-induced edema. In the present study, these results supported the previous findings that aucubin is a specific inhibitor of carrageenan induced hind paw edema, which might explain its beneficial effect in the treatment of inflammatory diseases. On the other hand, aucubin derivative and catalpol have no effect against

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carrageenan-induced edema, while ajugol was found to possess a weak activity in similar experimental protocols. A double bond between C-7 and C-8 is one of the most positive characters for activity, and its oxidation to an epoxy derivative leads to a remarkable decrease in anti-inflammatory activity, as occurs in catalpol (6). If an ester linking appears by acylation with an aromatic acid derivative at the sugar moiety, anti-inflammatory activity is augmented, whereas if acylation occurs directly in the monoterpenoid skeleton, this activity diminishes (Recio et al., 1994). However, in contrast with previous studies, our results showed that aucubin derivative (1) had no activity. Introduction of a hydroxyl function at C-8, as occurs in ajugol (7), decreases topical activity. The absence or presence of the extra-annual carboxyl or carboxymethyl group at C-4 is of no relevance, as is apparent result when the activity of aucubin is compared with those of geniposidic acid (5), but the conversion of a -COOH compound to its -COOMe derivative notably increases the topical activity (Recio et al., 1994). The potency ofilwensisaponin A was similar to that of indomethacin in acute anti-inflammatory experimental models and consistent, i.e. the effect was not reduced with time. It has recently been reported that other closely related saponins, verbascosaponin A and verbascosaponin, suppressed the edematous response 1 h after carrageenan injection, but this effect diminished slightly in due time (Giner et al., 2000). The mechanism of action, however, has not yet been established. It may be speculated that, the glucose molecule in the sugar residue connected to the C-3 position of the aglycone is crucial for its acute anti-inflammatory effect (Hostettmann & Marston, 1995; Gepdiremen et al., 2005). Outcome of the study, in connection with the role of aucubin and ilwensisaponin A as the active principles of V. lasianthum, it seems that they could be synergistic with other active substances in its fractions and extracts (Kupeli et al., 2007a). R1

OH

H OH

Rl 6-0-(4'" -O-trans-p-coumaroyl)H o:-L-rhamnopyranosylaucubin (2) 6-0-(4'" -O-trans-p- methoxy -cinnamoy1)- H 0:- L-rhamnopyranosylaucubin

(1)

(3) Sinuatol (4) Aucubin (5) Geniposidic acid

R2 (4'" -O-trans-p-coumaroyl)-o:-Lrhamnopyranose (4'" -O-trans-p-methoxycinnamoyl)-o:-Lrhamnopyranose 0:- L-rhamnopyranose

H H H COOH H

Fig 12. Contd.

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Effects of Medicinal Plants in Turkey Fig 12. Contd.

~

"~~~O" H~OH

OH

(6) Catalpol H06

~ 8

H

CH3

~ 1

0

~~OH H~OH

OH

(7) Ajugol

-~q

~q

YRO~O~

Me-r--o--1I'~ RO~ RO OR

OR I"

R

OR

0

~ RO

23

I"" OR

(8) Ilwensisaponin A Fig 12. Structures of the compounds (1-8)

In a different study, saponin glycosides called ilwensisaponin A and C and iridoid glycosides known as ajugol and picroside IV (Fig 13) were isolated from the Verbascum pterocalycinum var. mutense Hub.-Mor. methanolic extract. A dose-related anti-inflammatory and antinociceptive response were obtained in the study at doses of 100 and 200 mg/kg. The results of the evaluation of the anti-inflammatory activities induced by carrageenan and PGE 1 showed that this species possesses active constituents that could diminish cyclooxygenase activitiy. No effects were seen in the 12-0tetradecanoyl-13-acetate (TPA)-induced ear edema model. Ilwensisaponin A and C could explain in part the anti-inflammatory and analgesic activities of this species. Although antinociceptive and anti-inflammatory activities of ajugol and picroside IV were not significant in statistical analysis, ilwensisaponin A and C showed notable activity without inducing any apparent acute toxicity as well as gastric damage at 200 mg/kg dose (Kiipeli Akkol et al., 2007). In a reference survey, some triterpenes have been reported

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to have anti-inflammatory, cytotoxic, antitumor activities and to be chemopreventive. Papillomas in mouse skin were initiated with 390 nmol of7,12-dimethylbenz(a)anthracene and 1 week later, were promoted 2 times a week with 4.1 nmol of 12-0-tetradecanoylphorbol-13-acetate (TPA). Five saponin-related compounds used as potential anti-inflammatory agents from Verbascum songaricum were applied in the same position with 82 nmol. Saponin-related compounds effectively inhibited tumor formation in the sensitive mouse stock even when these compounds were given 1 h prior to TPA treatment. These results suggested that there was a general correlation between the anti-inflammatory and antitumor-promoting activities of saponins (Tokuda et al., 1991). Songarosaponins and their acylated derivatives, which had been previously isolated from Verbascum songaricum, have also been tested for anti-inflammatory activity using the croton oil ear model. Acylated derivatives showed excellent anti-inflammatory activity as compared to that of saponins (Anam, 2001). Giner et al. (2000) also demonstrated that verbascosaponin A and verbascosaponin, isolated from Scrophularia auriculata, significantly inhibited the mouse paw edema induced by carrageenan and ear edema induced by single and multiple doses ofTPA (Giner et al., 2000).

OH

O~O~O

H~ml~O HO

OH

~OH I"

HO H

OH

0

0

1m• OH

Ilwensisaponin C HO

7

o HO--'::::

O~O~O HO~OH OH

OH

o

Picroside IV Fig 13. Chemical structures ofisolated compounds

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Several Veronica species are reported to possess application in traditional medicines worldwide for the treatment of a wide range of disorders; as expectorant in respiratory diseases or as antiscorbutic, as diuretics and for wound healing (Baytop, 1999; Harput et al., 2002). In Chinese traditional medicine, V. anagallis-aquatica L. is used for the treatment of influenza, hemoptysis, laryngopharyngitis and hernia (Su et al., 1999). During our field expeditions on Turkish folk medicine, we have recorded that aerial parts ofV. anagallis-aquatica is boiled in milk to obtain poultice and then is applied on abdomen for abdominal pain or warm aqueous extract of leaves without removing the boiled herbs is used as a bath remedy to alleviate rheumatic pain in northwest Anatolia (Fujita et al., 1995). Previously, in vitro anti-inflammatory activity of five Veronica species (V. cymbalaria, V. hederifolia, V. pectinata var. glandulosa, V. persica and V. polita) were investigated through evaluation of the inhibitory effects on nitric oxide (NO) production in lipopolysaccharide-stimulated mouse peritoneal macrophages as well as cytotoxic activity against KB epidermoid carcinoma and B16 melanoma (Harput et al., 2002). MeOH extracts offive plant materials were found to possess inhibitory effects on NO synthesis in varying degrees. MeOH extracts were further partitioned between water and chloroform, and aqueous fractions showed the activity without inducing any cytotoxicity, while chloroform fractions were cytotoxic dose-dependently. Moreover, water fractions were found to possess remarkable effect on DPPH (2,2-diphenyl-1-picryl-hydrazyl), suggesting that the inhibitory effect on NO production might be due to their radical scavenging activity. In a study performed by our group, methanolic extract of the Veronica anagallisaquatica L. plant was shown to possess significant inhibitory activity against carrageenan-induced hind paw edema model and ofp-benzoquinone-induced writhings in mice. Through bioassay-guided fractionation and isolation procedures eight compounds, aquaticoside A (1), aquaticoside B (2), aquaticoside C (3), veronicoside (4), catalposide (5), verproside (6), verminoside (7) and martynoside (8) were isolated and their structures were elucidated by spectral techniques (Fig 14). Catapol derivative iridoid glucosides, catalposide (5) and verproside (6) were found to possess potent antinociceptive and anti-inflammatory activities at 500 mg/kg dose, per os without inducing any apparent acute toxicity as well as gastric damage (Kiipeli et al., 2005). Previously, anti-inflammatory effect of several iridoid derivatives has been reported by several other authors. Recio et al. (1994) studied the anti-inflammatory activity oftwelve iridoid glycosides against two inflammation models in mice, carrageenan-induced paw edema and TPA-induced mouse ear edema. Loganic acid and two catalpol derivatives, aucubin and 6' -vanilloylcatalpol, were found to possess potent inhibitory activity on the former, while catalpol derivatives, aucubin, verbenalin and loganin, were found remarkably active on the latter model. According to

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their experimental results, systemic anti-inflammatory activity ofiridoids was modest, while a higher efficacy could be observed through topic application as evidenced by TPA-edema model. They have further suggested a structure-activity relationship for topic anti-inflammatory activity of iridoids. Accordingly, OH-substitution as C 5 , unsaturation as C 7-C S' methyl substitution of carbonyl Cll' and integrity of the cyclopentane ring were essential for higher activity. Some of the results suggested in the study of Recio et al. (1994) were later supported by other authors as well; i.e. picroside II, a catalpol derivative, was found active only topically and exerted a moderate topic anti-inflammatory activity in mouse ear-swelling assay (Jia et al., 1999). This conclusion also supports the folkloric application of the plant in Turkey, where people applied boiled herbs ofV. anagallis-aquatica on their extremities, while taking bath in warm aqueous extract to alleviate rheumatic pain (Fujita et al., 1995). Furthermore, the conclusion suggested by Recio et al. (1994) reporting that a higher efficacy could be observed through topic application of iridoid glucosides than oral administration provides a further support to folkloric utilization in Turkey,

HO

o 1

10

0

I'

6'

3"

O-~o-R

~~H

-5"

OH R

H

(1) Aquaticoside A

OH

(2) Aquaticoside B

11

COOH

HO

o

0

3"

II~ -+-O~O-C~ °HO~OH -5" 1,

OH (3) Aquaticoside C

105

Effects of Medicinal Plants in Turkey Fig 14. Contd.

o I

I'

O~O~CH20H

HO~OH OH

R2

RI H H

H

(4) Veronicoside (5) Catalposide (6) Verproside

OH OH

OH

o II

7"

HO~" I" C-O

I

HO

'<:::::,

~4 5 ~3

'<:::::,8"

0

.#

5"

.

HOH2 /

9

8

10

0

OI~I' 0 CH20H OH

HO

OH

(7) Verminoside

~ HO

1'"

13'

~a'

~OH 4' 0

o 0

o

;:;H C--J-O--J I "

OO~ 3

HO

13

0H l'O~~ 3 a

3'

OH

~ 5

I

O~

OH (8) Martynoside

Fig 14. Chemical structures of isolated compounds 1-8

TAXACEAE

There are eight Taxus species and two hybrids in the world (Van Rozendall et al., 1999) and T. baccata L. (European yew) is the single representative in Turkey (Davis & Cullen, 1965). A large number of taxoids possessing different skeleton systems, as well as lignans, flavonoids, steroids and sugar derivatives were isolated from various Taxus species (Baloglu & Kingston, 1999; Parmar et al., 1999). During the course of studies on the bioactive

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components, the chloroform soluble portion of ethanolic extract of the heartwood ofT. baccata afforded four taxoids; taxusin, baccatin VI, baccatin III and 1b-hydroxybaccatin I, along with five lignans; lariciresinol, taxiresinol (3'-demethyllariciresinoD, 3'-demethylisolariciresinol-9'hydroxyisopropylether, isolariciresinol and 3-demethyl isolariciresinol (Erdemoglu, 1999). Lignans are known to possess various biological activities, including antibacterial, antifungal, antiviral, antioxidant, anticancer and anti-inflammatory effects (Cho et al., 2001a). As related with the abovepresented data, a study was designed to investigate in vivo anti-inflammatory and antinociceptive activity of lignans and taxoids isolated from the chloroform-soluble portion of ethanolic extract of the heartwood of T. baccata. All the isolated lignan derivatives and taxoid compounds by Erdemoglu et al. (1999) were shown to possess significant antinociceptive activity against p-benzoquinone induced abdominal contractions, while only lignan derivatives significantly inhibited carrageenan-induced hind paw edema in mice (Kiipeli et al., 2004). These results were in accordance with the previous studies. Two of the studied lignans; lariciresinol and isolariciresinol, were reported to possess potent in vitro inhibitory effect on TNF- production, a pro-inflammatory cytokine (Cho et al., 2001b); and thus lignan derivatives in the present study might show their anti-inflammatory effects via the same mode. On the contrary, the weak anti-inflammatory effect was observed for the taxoids studied in the present study, which might be ascribed to their TNF-inducing effect when stimulated with Salmonella LPS, as reported by Kirikae et al. (2000).

THYMELAEACEAE In Traditional Chinese Medicine, several Daphne species have been frequently added in formulations prescribed against inflammatory disorders; i.e. D. genkwa to dispel edema (Zheng et al., 2006), D. acutiloba for wounds and bruises (Taniguchi et al., 1998), D. tangutica for treatment of rheumatoid arthritis (Chen et al., 2004), seeds and barks, rarely leaves, of D. mezerium in chronic rheumatism, skin diseases, gout and inflammations in the lymph tissue (Chen et al., 2004). A piece of root ofthe latter species was also recommended to stop pain in toothache by chewing (Grieve & Leyel, 1967; Schildknecht et al., 1970). Aerial parts and roots of Daphne oleo ides ssp. oleoides are used for the treatment of rheumatic pain, lumbago, and to reduce fever in Turkish folk medicine. In Inner Anatolia, the fresh stem of the plant was employed to reduce swellings in the legs of cripple animals (Baytop, 1999; Yesilada et al., 2001). In an activity screening study on a number of plant extracts used for treatment of various inflammatory conditions, D. oleoides ssp. oleoides of Turkish origin was found to be the most effective against interleukin-1ex and TNF-ex (Yesilada et al., 1997). Then, through in vitro activity-guided fractionation procedures assessing the inhibitory effects on inflammatory cytokines two diterpenes and one

Effects of Medicinal Plants in Turkey

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coumarin derivative were isolated and chemically defined as the main principles that supported its the folkloric usage (Yesilada et al., 2001). In another study the in vivo anti-inflammatory and antinociceptive activities of D. pontica were studied. For this purpose, effects of extracts and fractions obtained through successive solvent extractions with n-hexane, diethyl ether, ethyl acetate and methanol from either the roots, leaves, stems or flowers with young leaves were investigated against carrageenan-induced hind paw edema, PGE 2-induced hind paw edema and 12-0-tetradecanoyl-13-acetate (TPA)-induced mouse ear edema models for the anti-inflammatory activity, and p-benzoquinone-induced abdominal constriction test for the antinociceptive activity assessment. Only ethyl acetate extracts ofthe roots showed significant anti-inflammatory activity on carrageenan-induced (22.732.0% inhibition) and PGE 2-induced hind paw edema (3.2-27.3% inhibition) as well as 12-0-tetradecanoyl-13-acetate (TPA)-induced mouse ear edema (47.8-43.3% inhibition) models at 50 mg/kg dose without inducing any apparent gastric lesion or acute toxicity, whereas the other extracts were shown to be ineffective. In addition to roots, ethyl acetate extracts of the stems also exhibited 19.5-29.9%; 5.3-23.9%; 36.6-28.1% inhibition on carrageenan-induced and PGE 2-induced hind paw edema as well as 12-0tetradecanoyl-13-acetate (TPA)-induced mouse ear edema models, respectively. On the other hand, none ofthe extracts showed antinociceptive activity (Kupeli et al., 2007b). TILIACEAE Silver linden, Tilia argentea Desf. ex DC. (Tiliaceae) [synonym: T. tomentosa Moench] inflorescence is a popular remedy worldwide against the symptoms of common cold, bronchitis and cough (Gruenwald et al., 2000b). Moreover, it is also claimed to be effective as a diaphoretic for feverish colds and infectious diseases where a sweating cure is needed as well as occasionally used as a stomachic, antispasmodic, diuretic and sedative, although these effects are notified to need experimental proof (Gruenwald et al., 2000). The use ofleaves as medicine is not common as that offlowers, but suggested to be employed as a diaphoretic, however the effect has not been evaluated scientifically so far. It is also reported that the Tilia flavonoids to possess some anti-edema effect, however the composition was not described (Gruenwald et al., 2000). In order to assess this information, antinociceptive and anti-inflammatory activity of the main two flavonoid glycosides; kaempferol-3, 7 -O-a-dirhamnoside and quercetin-3, 7 -O-a-dirhamnoside isolated from the leaves were investigated. Both compounds were shown to possess potent antinociceptive and anti-inflammatory activity in 50 mg! kg dose, per os, without inducing any apparent acute toxicity as well as gastric damage (Toker et al., 2004). The potent anti-inflammatory activity of different types of flavonoids was reported previously. The anti-

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inflammatory activity of quercetin was reported by Simoes et al. (1988) against carrageenan-induced paw edema model at a dose of20 mg/kg i.p. and was noted a more pronounced protective effect on the earlier stages of the oedematogenic response. It is suggested that some flavonoids including quercetin blocked both the cyclooxygenase and lipoxygenase pathways of the arachidonate cascade at relatively high concentrations, while at lower concentrations only the lipoxygenase pathway (Di Carlo et al., 1999). Actually these data were supported by the conclusion that quercetin is an effective inhibitor of phospholipase A2 which catalyses the hydrolysis of phospholipids to release arachidonic acid as the precursor of the inflammatory response (Harborne, 1994). Guardia et al. (2001) studied the anti-inflammatory activity of three flavonoids; quercetin (flavonol), hesperidin (flavanone glycoside) and rutin (quercetin rutinoside) in 80 mg! kg doses using an acute/subacute inflammation models. Accordingly, rutin was found very active in suppressing the edema, nodules and ankylosis as evidenced from arthrogram scores in subacute phase, while quercetin and hesperidin were shown to possess low activity. Rotelli et al. (2003) conducted an in vivo comparative study on the anti-inflammatory activity of various flavonoids (quercetin, rutin, morin, hesperetin and hesperidin) and found that quercetin (in 75 mg/kg dose) showed a remarkable inhibitory activity on carrageeenan-induced paw edema model on acute administration (between 55 to 66% inhibition) as well as on the chronic model against adjuvant-induced arthritis model, while rutin (in 150 mg/ kg dose) was only effective against the later. Sanchez de Medina et al. (2002) pointed out that quercitrin exerts anti-inflammatory activity in trinitrobenzene sulfonic acid-induced colitis with a narrow dose-response. As described above, quercitrin is also one of the flavonoid components of silver linden leaves. Matsuda et al. (2002) reported that the kaempferol glycosides (tiliroside and astragalin) to possess potent inhibitory effect on TNF-production. However, there is no experimental study on the antiinflammatory activity of kaempferol glycosides. Detailed information on the absorption features offlavonoids have not yet to be available. Although it has been claimed that only the flavonoid aglycones be able to pass through the gut wall, this might be largely depends on the chemical structure of flavonoid (Di Carlo et al., 1999). However, once absorbed, flavonoids influence many biological functions including protein synthesis, cell proliferation differentiation and angiogenesis for the benefit of mankind (Di Carlo et al., 1999). If this hypothesis is correct, a-glycosidic bonds of the flavonoid glycosides 1 and 2 isolated by Toker et al. (2004) might be subjected to hydrolysis in the gut through a-glycosidase type enzymes to give aglycone then pass the gut wall to exert its anti-inflammatory and antinociceptive activity.

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VITACEAE The leaves of Vitis vinifera (vine leaves) have been traditionally used as a food in both fresh and brined forms in Turkey. The leaves, due to astringent and haemostatic properties, are used in the treatment of diarrhoea, haemorrhage and varicose veins, and the juice of leaves has been used as an eye bath (Felicio et al., 2001). In order to evalute the biological effects of vine leaves, extracts were submitted to bioassay models such as antioxidant activity [free radical scavenging activity (1,1-diphenyl-2-picrylhydrazyl, DPPH% assay), iron (III) reductive activity (reducing power activity assay), capacity of inhibition of linoleic acid peroxidation (ferric thiocyanate and thiobarbituric acid method)], antinociceptive activity (p-benzoquinoneinduced abdominal constriction test) and anti-inflammatory activity (carrageenan-induced hind paw oedema model). All the vine leaf extracts were able to scavenge DPPH% radicals at physiological pH and did so in a concentration-dependent fashion as well as reduced iron (III) to iron (II). However none of the extracts was as effective as the positive controls ascorbic acid, BHT, and gallic acid. The aqueous extract prepared from fresh leaves showed moderate inhibition at 100 mg/kg dose on carrageenaninduced hind paw oedema and p-benzoquinone-induced abdominal constriction tests without inducing any gastric damage. The content of phenolic compounds in the extracts were also determined and hydroxycinnamic acids (e.g. caffeic acid), and flavonoids (e.g. quercetin) were verified by a reverse phase HPLC-PDA method in the extracts (Ko~ar et al., 2007).

CONCLUSIONS Ethnobotanical and ethnopharmacological surveys conducted by our research group have explicitly revealed that Turkish traditional medicines provide a vast source for the discovery of new leads to develop effective and safety agents for the treatment of diverse diseases. Detailed and programmed investigations on this subject along with clinical evaluation, derivatization and formulation studies may yield efficient products for the benefit of human health.

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5 Comparative Study of the Antigenotoxic Effects of Some Selected Natural Plant Products Against Methylmethane Sulphonate Induced Genotoxic Damage in Cultured Mammalian Cells YASIR HAsAN SIDDIQUE 1,* AND MOHAMMAD AFZAL 1 ABSTRACT In the present study the effect of ascorbic acid (20, 40 and 80 pM), nordihydroguaiaretic acid (NDGA) (O.S, 1.0 and 1.S pM) and apigenin (S, 10 and 20 pM) was studied against the genotoxic damage induced by 60 pM of methylmethane sulphonate on human lymphocytes culture using chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs) as a parameter. The treatments result in a dose dependent decrease in the CAs and SCEs, suggesting a protective role during the chemotherapy of cancer patients, as there are chances of developing secondary tumors. Ascorbic acid was most effective in reducing the genotoxic damage as compared to NDGA and apigenin, and apigenin was more effective as compared to NDGA i.e. Ascorbic acid> Apigenin> NDGA. The results of the present study suggest that ascorbic acid, NDGA and apigenin can modulate the genotoxicity of MMS in human lymphocytes in vitro. Key words : Ascorbic acid, nordihydroguaiaretic acid, apigenin, chromosomal aberrations, sister chromatid exchanges

INTRODUCTION The herbal preparations are traditionally used for the treatment of various diseases and for that reason it becomes necessary to assess the mutagenic 1. Human Genetics and Toxicology Laboratory, Section of Genetics, Department of Zoology, Faculty of Life Sciences, Aligarh Muslim University, Aligarh - 202 002, U.P., India. * Corresponding author: E-mail: [email protected]

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potential or modulating action of natural plant products when associated with other substances. Herbal medicine is the root of various traditional medicine systems around the world. About 25% of the crude drugs are directly obtained from the botanicals and another 25% derived from chemically altered natural products. Various traditional medicine systems around the world, including ancient Chinese medical system, Indian medicinal system and Amazonial ethnomedicine, rely heavily on herbs for health preservation (Jain et al., 2008). Despite the advances in pharmacology and synthetic organic chemistry the dependence on natural plant products remain unchanged (Fostar, 1999). Apigenin is one of the several active ingredients found naturally in many fruits and vegetables, including apples and celery. It is found in several popular spices, including basil oregano, tarragon, cilantro and parsley. A high amount of apigenin is found in parsley, peppermint, lemon, berries and fruits (Peterson & Dwyer, 1998). It is a member of flavone family of the flavonoid. Apigenin is recognized in traditional or alternative medicine for its pharmacological activity (Hoftman, 2000). In several strains of Salmonella typhimurium it was found negative for reverse mutation assay (NLM, 2000). It induced SOS repair system in E. coli K-12 stain in PQ37 with and without metabolic activation (Czeczot & Bilbin, 1991). Apigenin has also been reported to have anti-inflammatory effects, free radical scavenger, anticarcinogenic (Kim et al., 1998), grown inhibitor in various cancer lines (Yim et al., 2001; Wang et al., 2000) and antigenotoxic properties (Siddique et al., 2008; Khan et al., 2006). N ordihydroguaiaretic acid (NDGA) is a phenolic lignan, present in the evergreen shrubs, Larrea divaricata and Guaiacam officinale (Agarwal et al., 1991). NDGA possesses a number of interesting biological properties, that are of potential use for humans such as enzyme inhibitor (Capdevilla et al., 1988), antimicrobial (Hurtado et al., 1979), a protector from neurotoxicity and bladder toxicity (Frazier & Kehrer, 1993), stimulator of corpus luteum for the secretion of progesterone (Carlson et al., 1995), potential vas and branchiodialator (Nagano et al., 1996), antimutagenic action against alkylating drugs (Wang et al., 1991), synthetic progestins (Siddique et al., 2006; Siddique et al., 2007) and estrogens (Siddique & Mzal, 2004). Ascorbic acid is a powerful antioxidant, which plays an important role in intra cellular oxidation/reduction systems, and in binding of free radicals produced endogenously (Laurence et al., 1997). Methylmethane sulphonate is a monofunctional alkylating agent with both neoplastic and

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mutagenic activities (Mittal & Musarrat, 1990). It alkylates DNA at the N7 position of guanine and the N-3 position of adenine. All these changes may give rise to abnonnal base pairing at DNA replication (Madrigal-Bujaidar et al., 1998). The present study deals with the comparative study of ascorbic acid, NDGA and apigenin against the genotoxic damage induced by methylmethane sulphonate in cultured human lymphocytes.

MATERIALS AND METHODS Chemicals Apigenin (Sigma), NDGA (Sigma), Ascorbic acid (SRL); PhytohaemagglutininM, antibiotic-antimycotic mixture, RPMI-1640 (Gibco); dimethylsulphoxide, 5-Bromo 2-deoxyuridine, colchicine (SRL, India); Giemsa stain (Merk).

Human Lymphocyte Culture Duplicate peripheral blood culture were perfonned according to Carballo et al. (1993). Briefly, heparinized blood samples (0.5 mL), were obtained from a healthy female donor and were placed in a sterile culture tubes containing 7 mL ofRPMI-1640 medium, supplemented with fetal calf serum (1.5 mL), antibiotic-antimycotic mixture (1.0 mL) and phytohaemagglutinin CO.1 mL). The flasks were kept in an incubator at 37°C for 24 h.

Chromosomal Aberration Analysis After 24 h, 60 ~M of methylmethane sulphonate was given with 20, 40 and 80 ~m of ascorbic acid, 0.5, 1.0 and 1.5 ~M of NDGA and 5, 10 and 20 ~M of apigenin separately and kept for another 48 h. The treatment of selected doses ofMMS, ascorbic acid, NDGA and apigenin were also given separately. After 47 h, 0.2 mL of colchicine (0.2 Ilg/mL) was added to the culture flask. Cells were centrifuged at 1000 rpm for 10 min. The supernatant was removed and 5 mL of prewarmed (37°C) KCI hypotonic solution (0.075 M) was added. Cells were resuspended and incubated at 37°C for 15 min. The supernatant was removed by centrifugation at 1000 rpm for 10 min., and 5 mL of chilled fixative (methanol: glacial acetic acid; 3:1) was added. The fixative was removed by centrifugation and the procedure was repeated twice. The slides were stained in 3% Giemsa solution in phosphate buffer (pH 6.8) for 15 min. 300 metaphases were examined for the occurrence of different type of abnormality. Criteria to classify the different types of aberrations were in accordance with recommendation ofEHC 46 for environmental monitoring of human population OPCS, 1985).

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Sister Chromatid Exchanges Analysis For sister chromatid exchange analysis, bromodeoxy uridine (10 ~g/mL) was added at the beginning of the culture. Mter 24 h, 60 ~M of MMS treatment was given with 20, 40 and 80 ~ of ascorbic acid, 0.5, 1.0 and 1.5 ~M of NDGA and 5, 10 and 20 ~M of apigenin separately and kept for another 48 h in an incubator. Mitotic arrest was performed by adding 0.2 mL of colchicine (0.2 ~g/mL). Hypotonic treatment and fixation were performed in the same way as for the chromosomal aberration analysis. The slides were processed according to Perry and Wolff (1970). The sister chromatid exchange average was taken from an analysis of metaphase during second cycle of division.

Statistical Analysis Students t-test was used for the analysis of CAs and SCEs. Regression analysis was also performed for the dose effect of ascorbic acid NDGA and apigenin against the genotoxic dose of MMS using statistical software (Statsoft Inc.).

RESULTS The results of the present study reveal that the treatment of ascorbic acid, NDGA and apigenin reduced the genotoxic effect ofMMS. The used dosage of ascorbic acid, NDGA and apigenin were not genotoxic per se. The treatment of 20, 40 and 80 ~M of ascorbic acid, 0.5, 1.0 and 1.5 ~M of NDGA and 5, 10 and 15 ~M of apigenin with 60 ~ of MMS results in a dose dependent decrease in number of abnormal metaphases (Table 1). The chromatid exchanges and dicentric chromosomes were completely eliminated even at the lowest tested doses of ascorbic acid, nordihydroguaiaretic acid and apigenin (Table 1). In sister chromatid exchange analysis the treatment of 20,40 and 80 ~ of ascorbic acid, 0.5, 1.0 and 1.5 ~M of NDGA and 0.5, 10 and 15 ~M of apigenin with 60 ~M of MMS results in a dose dependent decrease in SCEs/cells (Table 2). Regression analysis was also performed to determine the dose of effect of ascorbic acid, NDGA and apigenin on 60 ~ ofMMS for number of abnormal meta phases and sister chromatid exchanges. A decrease in the slope of linear regression was observed as the dose of ascorbic acid, NDGA and apigenin was increased. For abnormal metaphases the treatment of 20, 40 and 80 ~M of ascorbic acid (F = 3.85; p<0.10; Fig 1), 0.5, 1.0 and 1.5 ~M of NDGA (F = 27.0; p<0.02; Fig 2) ad 5, 10 and 20 ~M of apigenin (F = 7.68; p<0.08; Fig 3) with the increase in the dosage a decrease in the slope of linear regression lines was observed. For sister chromatid exchange analysis, the treatment of 20, 40 and 80 ~M of ascorbic acid (F = 6.57; p<0.063; Fig 4),0.5, 1.0 and 1.5 ~M ofNDGA (F = 30.37; p<0.02; Fig 5) and

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5, 10 and 20 J.1M of apigenin (F =24.14; p<0.024; Fig 6) with the increase in the dosage the decrease in the slope of linear regression lines was observed Table 1. Effects of ascorbic acid, nordihydroguaiaretic acid and apigenin on chromosomal aberrations induced by methylmethane sulphonate Treatments

MMS(IIM) 60 MMS(IJM)+ AA(IJM) 60+ 20 60+40 60+80 MMS(IJM)+ NDGA (IJM) 60 + 0.5 60 + 1.0 60 + 1.5 MMS (IJM) + Apigenin (11M) 60+5 60+ 10 60 + 20 AA(IJM) 20 40 80 NDGA (IJM) 0.5 1.0 1.5 Apigenin (IJM) 5 10 20 Untreated Negative control (DMSO, 51l1/mL)

Chromosomal aberrations

Abnormal metaphases without gaps Number

Mean%:tSE

Gaps

CTB

CSB

CTE

DIC

38

12.67 ± 1.92"

23

22

16

5

4

18 12 10

6.00 ± 1.37b 4.00 ± l.13b 3.33 ± 1.03b

12 8 6

10 8 7

8 4 3

28 24 22

9.33 ± 1.67b 8.00 ± 1.56b 7.33 ± 1.50b

16 14 10

16 14 13

14 12 9

23 16 12

7.67 ± 1.53b 5.33 ± 1.29b 4.00± 1.13b

9 8 5

13 9 7

10 7 5

2 3 4

0.67 ± 0.47 1.00 ± 0.57 1.33 ± 0.66

1 2 2

2 2 2

1 2

4 5 6

1.33 ± 0.66 1.67 ± 0.73 2.00 ± 0.80

2 2 3

2 3 3

2 2 3

2 3 4 2 2

0.67 ± 0.47 1.00 ± 0.57 1.33 ± 0.66 0.67 ± 0.47 0.67 ± 0.47

1 2 2 1 1

2 2 2 2 2

1 2

CTB: Chromatid break; CSB: Chromosome break; CTE: Chromatid exchange; DIC: Dicentric; MMS: Methylmethane sulphonate; AA: Ascorbic acid; NDGA: Nordihydroguaiaretic acid; DMSO: Dimethylsulphoxide; SE: Standard error. aSignificant with respect to untreated (p
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Table 2. Effects of ascorbic acid, nordihydroguaiaretic acid and apigenin on chromosomal aberrations induced by methylmethane sulphonate SeEs/cell (mean :t SE)

Treatments MMS(J1.M) 60 MMS (J1M) + AA (J1M) 60 + 20 60 +40 60 + 80 MMS (J1M) + NDGA (J1M) 60 + 0.5 60 + 1.0 60 + 1.5 MMS (J1M) + Apigenin (J1M) 60+ 5 60 + 10 60 + 20 AA (J1.M) 20 40 80 NDGA (J1.M) 0.5 1.0 1.5 Apigenin (J1.M) 5 10 20 Untreated Negative control (DMSO, 5 JllImL)

17.22 ± 0.98a 13.24 ± 0.74b 10.34 ± 0.68b 8.84 ± 0.59b 14.12 ± O.84b 12.28 ± 0.76b 11.32 ± 0.74b 13.68 ± 0.78b 12.02 ± 0.72b 10.42 ± 0.70b 2.14 ± 0.27 2.36 ± 0.32 2.94 ± 0.36 2.56 ± 0.34 3.22 ± 0.40 3.98 ± 0.46 1.72 ± 1.76 ± 1.80 ± 1.52 ± 1.58 ±

0.28 0.30 0.31 0.20 0.26

MMS: Methylmethane sulphonate; AA: Ascorbic acid; NDGA: Nordihydroguaiaretic acid; DMSO: Dimethylsulphoxide; SE: Standard error. aSignificant with respect to untreated (p
9 10

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20

30

40

50

60

70

60

80

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Concentration of apigenin

Fig 1. Effect of ascorbic acid on number of abnormal metaphases induced by 60 J1M of methylmethane sulphonate

127

Antigenotoxic Effects of Some Selected Natural Plant Products Y=30657-6.0OO·X Correlation: r = -.9820

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Y = 13.990 - .0682' X Correlation: r = -.9317 14.5,------------------------------,

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Fig 4. Effect of ascorbic acid on sister chromatid exchanges induced by 60 IlM of methylmethane sulphonate

Compo Bio. Nat. Prod., Vol. 2 -Efficacy, Safety & Clinical Evaluation I

128

Y =15.373 - 2.800· X Correlation: r = -.9839 14.6

1'"

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Fig 5. Effect ofnordihydroguaiaretic acid on sister chromatid exchanges induced by 60 J.lM of methylmethane sulphonate

=

Y 14.480 - .2091 • X Correlation: r = -.9799

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Fig 6. Effect of apigenin on sister chromatid exchanges induced by 60 IlM of methylmethane sulphonate

DISCUSSION The results of the present study reveals that all the natural plant product are effective in reducing the genotoxic damage of methylmethane sulphate. Ascorbic acid is most effective in reducing the genotoxic damage as compared to apigenin and NDGA. In our present study the following trend was found in the potency of reducing the genotoxic damage induced by methylmethane sulphonate-Ascorbic acid> Apigenin> N ordihydroguaiaretic acid. Vitamins act as antioxidants and free radical scavengers, thereby acting as anticarcinogenic, anticlastogenic and antimutagenic agents. Ascorbic acid

Antigenotoxic Effects of Some Selected Natural Plant Products

129

possesses a substantial nucleophilic character and it has been suggested that ascorbate might protect against electrophilic attack or cellular DNA by intercepting reactive agents (Edgar, 1974) or that ascorbyl anion radical with a high extent of unpaired electron delocalization is responsible for the scavenging of free radicals (Bielski, 1961; Szeto et al., 2002). As the treatment with ascorbic acid results in a decrease of the genotoxic damage induced by MMS, it may be due to the scavenging property of ascorbic acid. NDGA possesses antioxidant (Olivetto, 1972) and free radical scavenging property (Nakadate, 1989; Marthy et al., 1990), which may be useful in reducing the genotoxic damage induced by MMS. The doses ofNDGA were selected on the basis of study performed by Madrigal-Bujaidar et al. (1998). Genotoxicity testing provides human a risk assessment. An increase in the frequency of chromosomal aberrations in peripheral blood lymphocytes is associated with an increased overall risk of cancer (Hagmar et al., 1984). Most of the chromosomal aberrations observed in the cells are lethal, but there are many other aberrations that are viable and cause genetic effects, either somatic or inherited. The ready quantifiable nature of sister chromatid exchanges with high sensitivity for revealing toxicant-DNA interaction and the demonstrated ability of genotoxic chemicals to induce significant increase in sister chromatid exchanges in cultured cells has resulted this endpoint being used as indicator of DNA damage in blood lymphocytes of individuals exposed to genotoxic carcinogens (Albertini et al., 2000). The above genotoxic endpoints are well known workers of genotoxicity of any reduction in the frequency of these genotoxic endpoints gives an indication of the antigenotoxicity of a particular compound (Albertini et al., 2000). Genotoxic effect of anti cancer drugs in non-tumor cells are of special significance due to the possibility that they may induces secondary tumors in cancer patients. Therefore, the antigenotoxic effects of these natural plant products are of special significance, that they are potent enough in reducing the genotoxic damage, thereby reducing the possibility of developing secondary tumors in the patients undergoing chemotherapy, but care should be taken with regard to their concentration if consumed in isolation.

ACKNOWLEDGEMENTS Thanks are due to DST, New delhi for the project No: SRIFTILS-003/2007 under SERC Fast Track Scheme for young scientists to the author Yasir Hasan Siddique and to the Chairman, Department of Zoology, AMU, Aligarh-202002 for the laboratory facilities.

REFERENCES Agarwal, R., Wang, Z.Y., Bik, D.P. and Mukhtar, H. (1991). Nordihydroguaiaretic acid, an inhibitor oflipoxygenase, also inhibits cytochrome P-450-mediated monoxygenase activity in rat epidermal and hepatic microsomes. Drug Metabolism Disposition, 19: 620-624. Albertini, R.J., Anderson, D., Douglas, G.R., Hagmar, L., Heminiki, K., Merelo, F., Natrajan, A.T., Norppa, H., Shuker, D.E.G., Tice, R., Walters, M.D. and Aitio, A. (2000). IPeS

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guidelines for the monitoring of genotoxic effects of carcinogens in humans. Mutation Research, 463: 111-172. Bielski, AH.J., Allen, O. and Schwarz, H.A (1981). Mechanism of disproportion action of ascorbic radicals. Journal of American Chemistry, 103: 3516. Capdevilla, J., Gil, L., Orellana, M., Marmett, 1.J., Mason, J.L., Yadagiri, P. and Falck, J.R (1988). Inhibitor of cytochrome P-450 dependent arachidonic acid metabolism. Archives of Biochemistry and Biophysics, 261: 257-263. Carlson, J.C., Sawada, M., Boone, D.L. and Stauffer, J.M. (1995). Stimulation of progesterone secretion in dispersed cells of rat Corpora lutea by antioxidants. Steroids, 60: 272-276. Czeczot, H. and Bilbin, M. (1991). Effect of flavones and their metabolites on induction of SOS repair in the strain PQ37-E.coli K-R Acta Biochimica Polonica, 38: 71-74. Edgar, J.A (1974). Ascorbic acid and biological alkylating agents. Nature, 248: 136-137. Foster, S. (1999). Medicinal plant development in the United States. In: Special Forest Products: Biodiversity meets the market place (General Technical Report GTR-WO-63). Ed. by Vance, N.C. and Thomas, J., Washington, DC: USA Forest Service. Frasier, L. and Kehrer, J.P. (1993). Effect of indomethacin, aspirin, nordihydroguaiaretic acid, and piperonyl butoxide on cyclophosphamide induced bladder damage. Drug Chemical Toxicology, 16: 117-133. Hagmar, L., Bonassi, S., Stromberg, V., Brogger, A., Knudson, J.E., Norppa, H. and Reuterwall, C. (1998). Chromosomal aberrations in human lymphocytes predict human cancer: A report from the European study group on cytogenetic biomarkers and health (ESCH). Cancer Research, 58: 4117-4121. Hoftman, D.L. (2000). Chamomile, Herbal Materia Medica < http://www.inpicentor.com/ librarylherballmaterial-mediallchamomile.asp>. Hurtado, L., Hernandez, R, Hernandez, F. and Fernandez, F. (1979). Fungitoxic compound in the Larrea resin. In: Centro de Investigacion en Qumija Aplicada Ed. by Campos, E., Mabri, T.J. and Fernandez, S., Mexico, pp. 328-340. Jain, A, Porwal, P., Jain, AK. and Bhargava, S. (2003). Arthritis and Herbs, In: Recent Progress in Medicinal Plants, Vol. 19, Ed. by Singh, V.K., Govil, J.N. and Sharma, RK., Studium Press LLC, USA, pp. 91-105. Khan, T.H., Jahangir, T., Prasad, L. and Sarwat, S. (2006). Inhibitory effect of apigenin an benzo (a) pyrene mediated genotoxicity in Swiss albino mice. Journal of Pharmacy and Pharmacology, 58: 1655-1660. Kim, H.P., Mani, 1., Iversen, L. and Ziboh, V.A. (1998). Effect of naturally occurring flavonoids and bioflavonoids on epidermal cycloxygenase and lipoxygenase from guinea pigs. Prosta glandons Leukot Essential Fatty acids, 58: 17-24. Laurence, D.R., Dennett, P.N. and Brown, M.J. (1997). Clinical Pharmacology, eighth Edition, New York, Churchill Living Stone. Madrigal-Bujaidar, E., Diaz Barriga, S., Cassani, M., Marquez, P. and Revuelta, P. (1998). In vivo and in vitro antigenotoxic effect nordihydroguaiaretic acid against SCEs induced by methylmethane sulphonate. Mutation Research, 419: 163-168. Marthy, S.N., Cooney, C.G. and Clearfield, H.R. (1990). Hydrogen peroxide-induced alterations in prostaglandins secretion in the rat colon in vitro. Inflammation, 14: 645-661. Nagano, N., Imaizuni, Y., Hirano, M. and Watanabe, M. (1996). Opening of Ca 2+-dependent K+ channels by nordihydroguaiaretic acid in porcine coronary arterial snoop muscles cell. Japanese Journal of Pharmacology, 70: 281-284. Nakadate, T. (1989). The mechanism of skin tumor promotion caused by phorbal esters: possible involvement of arachidonic acid cascade/lipoxygenase, protein kinase C and calcium/calmodulin systems. Japanese Journal of Pharmacology, 49: 1-9. NLM (2000). CCRIS (Chemical Carcinogenesis Research Information System), National Library of Medicine, Bethesda, MD (Record Nos. 3789, 3790). Olivetta, E.P. (1972). Nordihydroguaiaretic acid. A naturally occurring antioxidant. Chemical Industry, 2: 677-679. Peterson, J. and Dwyer, J. (1998). Flavonoids: dietary occurrence and biochemical activity. Nutrition Research, 18: 1995-2018.

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Rithidech, K.N., Tungjai, M. and Whorton, E.B. (2005). Protective effect of apigenin on radiation induced chromosomal damage in human lymphocytes. Mutation Research, 585: 96-106. Siddique, Y.H. and Afzal, M. (2004). Antigenotoxic effect of nordihydroguaiaretic acid (NDGA) against SCEs induced by estradiol-17~ in human lymphocyte chromosomes in vitro. Indian Biologist, 36: 25-27. Siddique, Y.H., Ara, G., Beg, T. and Afzal, M. (2007). Protective role of nordihydroguaiaretic acid (NDGA) against the genotoxic damage induced by ethynodioldiacetate in human lymphocytes in vitro. Journal of Environmental Biology, 28: 279-282. Siddique, Y.H., Beg, T. and Afzal, M. (2006). Protective effect of nordihydroguaiaretic acid (NDGA) against norgestrel induced genotoxic damage. Toxicology in Vitro, 20: 227-233. Siddique, Y.H., Beg, T. and Afzal, M. (2008). Antigenotoxic effect of apigenin against anticancerous drugs. Toxlcol in Vitro, 22: 625-63l. Snyder, RP. and Gillies, P.J. (2002). Evaluation of the clastogenic, DNA intercalation, and topoisomerase II interactive properties of bioflavonoids in Chinese Hamster V79 cells. Environmental and Molecular Mutagenesis, 40: 266-276. Szeto, Y.T., Tomlinson, D. and Benzie, I.F.F. (2002). Total antioxidant and ascorbic acid content of fresh fruits and vegetables: implications for dietary planning and food preservation. British Journal of NutritIOn, 87: 55-59. Wang, W., Heideman, L., Chung, C.S., Pelling, J.C., Koehler, K.J. and Birt, D.F. (2000). Cell cycle arrest of G21M and growth inhibition by apigenin in human colon carcinoma cells lines. Molecular Carcmogenesis, 50: 102-110. Wang, Z.Y., Agarwal, R., Zhou, Z.C. and Bickers, D.R (1991). Anti-mutagenic and antitumorigenic activities of nordihydroguaiaretic acid. Mutation Research, 261: 155-162. Yin, F., Giuliano, A.E., Law, RE. and Van Herle, A.J. (2001). Apigenin inhibits growth and induces G21M arrest by modulating cyclin-cdk regulators and ERK-MAP kinase activation in breast carcinoma cells. Anti Cancer Research, 21: 413-420.

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6 Inflammation and Mediators S. SINGH 1,*,

P.

A. KOULl, R. SHARMA1, G.D. SINGHl, A. KIiAJuRIAl AND V.K. GUPTAl

KOULl,

ABSTRACT Inflammation is the normal protective response to tissue injury caused by any noxious stimulus to destroy the invading organisms, remove irritants and set stage for tissue repair. It is triggered by the release of different cellular and humoral mediators from the injured tissues and migrating cells. The specific mediators vary with the type of inflammatory process. Enormous numbers of them are known today. It has been said that medical knowledge doubles every 10 years. With this assumption, if we start with one mediator in the early twenties we end up with a very high number till today. The concept of inflammatory mediators may sound reasonably limited yet the field has become so specific that no single individual is known who masters it all. As the number of mediators has grown, so has the number of targets. Unti/1930's the microcirculation attracted all the attention, in late 30's existence of accumulation of leukocytes in terms of mediators i.e., endogenous substances capable of attracting white blood cells out of the blood vessels. 70's was the era of prostaglandins which created revolution in the history of inflammation and ended with COX-1 and COX-2. Today any cell is a fair target for mediators. The functional response may be extremely subtle but a cell can be induced to express or reveal a new antigen on its surface without any visible change or the target cell simply becomes activated. It is well known that all inflammatory cells can exist in these two conditions - resting and activated - and this may ultimately become true for all cells. Activated cells then produce new mediators of their own and the situation become more complicated. An attempt has been made in this review 1. Indian Institute ofIntegrative Medicine, Canal Road, Jammu Tawi - 180001, India. Corresponding author: E-mail: [email protected]

*

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to explain some of the hidden facts of inflammation through explaining the mediators of inflammation. Key words : Inflammation, mediators, cells, prostaglandins, cytokines, leukotrienes, selectins, integrins INTRODUCTION Ever since the dawn of history, man has always tuned to plants for alleviation of his sufferings. Herbal drugs have been used in the treatment of diseases since time immemorial. Written history on the use of medicinal and aromatic plants can be traced back to about 1000 years. Four fifth of the population in the world lives in the developing countries where majority of the people benefit from traditional medicines and only 15% ofthem benefit from allopathic medicines. Asia is the most important continent for the traditional medicinal plants. India, being an agricultural country, is richly endowed with a wide variety of indigenous floras, some of which have long been employed for their medicinal use. Several indigenous systems of medicines are practiced in India such as Ayurveda, Unani, and Siddha etc. All of them use a variety of herbs, minerals and other biological products as medicaments since long. These natural products are used in traditional medicines on the basis of subtle principles and concepts. These principles are basically similar in all the indigenous systems of Ayurveda, Unani, and Siddha. These are vividly described in Ayurvedic classics as a series oftexts called "nighantus" especially devoted to the usage of herbal drugs and their properties. The recent upsurge of global interest in herbal drugs has created a fresh impetus on the development of plant-based medicines and health products.

As science progresses, the unknown diseases are being discovered and reported. The diseases, which were rarely heard of, are becoming very common. With the advancement in the drug development system, many new drugs are flooding the world market but still they have not succeeded over the drudgery of diseases. Uncounted number of dreadful diseases has been listed in the medical history; inflammation is one among them and a concerned disease particularly rheumatoid arthritis. Although, inflammatory diseases have affiicted a large proportion of world population thus crippling the activity of human beings without killing, yet, no permanent cure. Every individual, a human or an animal must defend itself against multitude of different pathogens including viruses, bacteria, fungi, protozoan, metazoan parasites as well as tumours and a number of various harmful agents which are capable to derange its homeostasis. Inflammation is a complex stereotypical body's reaction to invasion by any infectious agent, antigen challenge or even just physical or traumatic damage.

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To talk of inflammation, war is the perfect metaphor for it. Both are necessary evils, more-or-Iess displaying stereotyped responses to outside threats. The specialized troops (white cells), suicide-commandos (neutrophils), long term siege armies (granulomas), supply routes (vessels), communications and intelligence (mediators), and a huge array of lethal weapons (inflammatory enzymes) come to patrolling the site of inflammation and surmount an inflammatory response. Despite idealistic rhetoric about "the laws of war", when the fighting starts, there is really only one law: ''kill your enemy". Like it or not, if you want peace, you must be prepared to fight under certain conditions. If you want to be healthy, your body must be able to mount an inflammatory response. Force will always rule our world. Our best hope is that this will be the force of good laws. And the best for which we can hope from the inflammatory response is that, for most of our lives, it will do us more good than harm. "Big Robbins" defines "inflammation as the reaction of vascularized living tissue to local injury". Inflammation is the name given to the more or less stereotyped ways our tissues respond to noxious stimuli, with blood vessels and white blood cells as its twin center pieces and a host of proteins as actors. Inflammation destroys, dilutes, or walls off the injurious agents and sets in motion the limited powers of the body to heal it. The main purpose of inflammation is to bring fluid, proteins and cells from the blood into the damaged tissue for the defense. In other words inflammation, a localised protective event, elicited by injury, is a complex and multimediated process. It may be considered as an essential protective and normal process to any noxious stimulus that may threaten the well being of the host ranging from a transient, self limiting and localised to a complex sustained response involving a part or the whole organism that may lead to the immunological rejection of foreign tissue. When tissue injury is caused by a single finite event, the inflammation and reparative process progresses smoothly from injury to healing. In these circumstances, the whole inflammatory process is truly beneficial and provides an example of homeostatic mechanism restoring the affected tissue to its former normal healthy state. In contrast, when the injurious agent is self replicating parasite, the inflammatory response becomes much more complex because some form of tissue injury will continue till the agent persists. Living tissue's early local response to an injury is acute inflammation, whereas, if acute inflammation persists for longer time it turns to chronic inflammation which is often systemic and biochemically different from acute inflammation, meaning thereby that inflammation can be acute or chronic depending upon the injurious agents, although acute and chronic inflammatory events often occur simultaneously. The physiological features of inflammation have been recognised since Celsus described them in first century A.D. Acute inflammation is

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recognised by cardinal signs of redness, oedema, heat, pain and loss of function. In fact acute inflammation is not a disease, but a defensive response to injury; however, it is not always beneficial. Acute inflammation comprises the immediate and early response to an injurious agent. The events in the acute inflammatory process are largely mediated by the production and release of a variety of chemical mediators. Despite the diversity, the injurious influence and the tissue involved in inflammation, similar chemical mediators are released, and hence the acute inflammatory process is virtually stereotyped. Further more, the acute inflammation may remain confined to the site of injury and evoke only local signs and symptoms or may be extensive and induce systemic signs as well as evolve secondary line of defense, such as lymphoid tissue. The protein rich fluid and white cells usually polymorphs, migrate from blood that accumulate in the extravascular spaces as a result of an inflammatory reaction, constitute an exudate indicating that acute inflammatory reactions are exudative, while on the other hand chronic inflammation is largely a proliferative one. Furthermore, inflammatory process has been described in terms of the immediate reaction to injury, which gives rise to acute inflammation. The acute response is characterised principally by the vascular and exudative changes. The white cells that participate in acute inflammation are usually neutrophils and macrophages. In contrast, chronic inflammation results from injurious stimuli that are persistent, often for weeks or months leading to a predominating proliferative rather than an exudative reaction. Chronic inflammatory reactions are also characterized by white cell accumulations, chiefly macrophages and lymphocytes and some times plasma cells. Thus, leucocytic exudate in chronic inflammation is mononuclear, whereas in acute inflammation it is polymorphonuclear. Chronic inflammation may arise following acute inflammation or the response may be chronic almost from the onset. Acute to chronic transition occurs when the acute inflammatory response cannot be resolved, due to either the persistence of the injurious agent or to some interference in the normal process of healing. Chronic inflammation is associated with a range of inflammatory diseases, such as rheumatoid arthritis, hepatitis, chronic pulmonary disease, various inflammatory diseases of gastrointestinal tract and chronic periodontal diseases. Chronic inflammatory diseases show a pathological immune response probably due to the presence of perpetuating antigenic determination. Under certain conditions immune reactions are set up against the individual's own tissue, leading to autoimmune diseases. In these diseases auto antigens evoke a self perpetuating immunological reaction that results in several chronic inflammatory diseases, such as rheumatoid arthritis and chronic thyroiditis.

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Some inflammations are difficult to categorise as acute or chronic because there is no sharp line that divides them. Arbitrarily it is said that when an inflammation lasts more than four to six weeks it is said to be a chronic inflammation. However, much depends on the effectiveness of the host response and nature of the injury. Time limits are without meaning. The differentiation of acute and chronic inflammation can best be made on the basis of morphological pattern of the reaction.

INFLAMMATION It is presumed that inflammation is a salutary process. It may have the injurious effects on the body. Teleologically, it is a process of self defenses against noxious stimulus to a certain limit, beyond that it requires drug therapy to curtail the limits of the process. Broadly inflammation is a complex cellular and biochemical response to injurious stimuli that involves a multitude cell types and mediators. It is classified into acute and chronic types. The account of acute and chronic inflammation often separates the two conditions by a description of healing and regeneration. An understanding of the histological picture of chronic inflammation is certainly assisted by knowledge of the process involved in repair, but the separation from acute inflammation leaves the impression that these two varieties of inflammation are distinct from each other, although sharing the term inflammation in their name. In general, the term acute, as applied to inflammation, refers to a response with a rapid onset and relatively short duration characterized by particular vascular phenomena. The common sequel is resolution of the inflammatory exudate and restoration of normal structure and function. In some cases the acute events lose their intensity, so that the process continues as a smoldering chronic condition. As long as irritation persists, the lesion should be considered inflammatory and once the irritation abates or is removed, the process becomes reparative.

Acute Inflammation The inflammatory process has been described in terms of the immediate reaction to injury which gives rise to acute inflammation. The physiological features of acute inflammation have been recognised since Celsus described them in the first century A.D. as the cardinal signs. These are redness, oedema, pain and heat. The fifth sign i.e., loss of function was added later by Galen (1978). Infact acute inflammation is not a disease, but a defensive response to injury, however, it is not always beneficial. In human beings inflammation occurs in vascular tissues, and is primarily a local vascular response where protein rich fluid and cells are brought to the site of injury to neutralize the damaging agent. The damaging agent can be due to a variety of causes viz., mechanical damage (cuts, blows etc.), chemical damage (acids, alkalies, toxins etc.), radiation, burns, scalds, viruses, bacteria, parasites, insect bites, stings, and local pathology such as antigen-

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antibody reactions. Early events in inflammation are obviously vascular in nature and these can be demonstrated on the skin by stricking it with a ruler. Initially a white line is produced because of mechanical stimulus to local capillaries, which have responded with vasoconstriction when blood is pushed out of the vessels. This is a transitory phenomenon, but if sufficient pressure is applied a red line results which is because of vasodilation where the new dilated capillaries fill with blood and produce the red color. The sharpness of the red line fades in about thirty seconds to one min and the area of pressure takes on a dull red, blotchy appearance. This is known as flare and is caused by arteriolar dilation. If sufficient force was applied in the initial trauma a wheal will develop due to the loss of fluid through the vascular wall, resulting in the formation of oedema. As the oedema developed some of the redness may be lost where pressure from the collected fluid close down the capillaries. Vasodilation, in particular, arteriolar dilation increases the local blood supply which, being at a higher temperature than surface tissue, gives a feeling of warmth. These vascular changes are due to chemical mediators being released at the site of injury. The early changes in the blood vessel wall and the changes in the blood flow are known as "stasis". As the fluid leaves the blood with the result it becomes more viscous and the flow slows down, the erythrocytes tend to stick together in the form of rouleaux and push the white cells towards the vessel walls. The collision of white cells with the vessel endothelium lining stimulates them and they increasingly adhere to the vessel wall and this adherence of cells to the vessel wall is known as pavementing of cell. Changes also occur in the endothelial cells of the vessel wall. A sticky film containing fibrin, which entraps polymorphonuclear leukocytes and other blood cells such as platelets, covers their surface. The passage of fluid through the blood vessel walls is due to changes in the hydrostatic and osmotic pressure. In acute inflammation the arteriolar dilation increases the flow of blood in the inflamed region and the pressure on small blood vessels. Eventually when the pressure in the capillaries exceeds the osmotic pressure of the proteins, fluid will leave the vessels and infiltrate into the tissues and form an exudate. Normal interstitial fluid has a low protein content and low specific gravity. In acute inflammation there are changes in the permeability of blood vessel wall, which allow both proteins and cells to pass through it. Although the passage of plasma proteins through the vessel wall is a passive process but cells leave by a different mechanism. The first cells to leave the blood vessels are the PMNLs, but if inflammation persists, monocytes follow and after some time become the dominant cells infiltrating into the inflamed tissue. Erythrocytes can also leave the blood vessels by a passive movement known as diapedesis.

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Mter they leave blood vessels, white cells show directional movement towards the injured tissue. About 90% of PMNLs are neutrophils. Infact the neutrophils are the typical cells of acute inflammation. In vitro neutrophils move faster than the monocytes but the neturophils are short lived, whilst monocytes and macrophages are comparatively long-lived cells. These factors may explain why neutrophils are predominant in acute inflammation, but monocytes, and macrophages and other cells such as plasma cells are predominant in established or chronic inflammations.

Chronic Inflammation Chronic inflammmation may arise following acute inflammation or the response may be chronic almost from the onset. Chronic inflammation usually occurs when acute inflammation remains unresolved due to either the persistence of the injurious agent or to some interference in the normal healing process. Chronic inflammation is associated with a range of inflammatory diseases such as rheumatoid arthritis, hepatitis, chronic obstructive pulmonary disease, various inflammatory diseases of gastrointestinal tract and chronic inflammatory periodonal disease. Chronic inflammatory disease shows a pathological immune response probably due to the presence of a perpetuating antigenic determination. Cells derived from three lines viz., mononuclear phagocyte, lymphoid cells and fibroblasts infiltrate the tissues. In addition there may be granulocytic leukocytes, particularly eosinophils. Macrophages, epitheloid cells, or multinucleated giant cells represent mononuclear phagocytes. Lymphoid cells are present as lymphocytes, plasma cells, or immunoblasts. Fibroblasts produce collagen and ground substance and proliferate to form more fibroblasts. At varying stages during the course of the lesion, lymphocytes, fibroblasts, or granulocytes may predominate, but the most important unit of chronic inflammation is almost always the macrophage and its derivatives, the epitheloid cells and the giant cells. Necrosis is also common in chronic inflammatory lesions. The overall participation of the dividing cells and their efforts in producing new tissue have resulted in chronic inflammation, being described as proliferative, in contrast to the exudative features of acute inflammation. In acute inflammation, macrophages disappear in a few days because of death of the cells, their removal by lymphocytes or migration elsewhere. In chronic inflammation the persistence of macrophages may result from their local proliferation, from longevity of the cells in the tissue, or from their continuing mobilization from the bone marrow. Although the mechanism of inflammation has been studied extensively, it is only recently that major attention has been paid to the target areas available for the pharmacological attack. Only a few drugs help to cure the disease. Many drugs assist the body's own healing process by holding a disease at bay, thereby allowing the nlltural defenses to build

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up or recover after the initial onslaught of disease. Other drugs such as non-steroidal antiinflammatory drugs merely relieve the symptoms of disease such as the pain and swelling in arthritis, without actually affecting the disease process. The major target areas identified for the pharmacological attack are as followed. 1.

Factors responsible for the onset of inflammation.

2.

Mediators released during initial injury

3.

Non specific endogenous antiflammatory factors evoked by the injured tissue.

4.

The processes attempting to restore normal function.

These target areas are inter-related as a chain reaction and the interruption of this chain at any point helps and lead into the success.

Biochemical Events A series of characteristic changes occur in the small blood vessels and the related tissues within the damaged area, when the living tissues are injured. The reaction in the first few hrs. is more or less independent of the nature of the injurious agent. The biochemical events that form the basis of acute inflammation are triggered by a number of compounds. The biochemical changes occur simultaneously and aid numerous changes. The different compounds with diverse chemical structure and actions aiding to inflammation are called inflammatory mediators, which are derived both from tissues and vascular components of the damaged area. Most of the cells release histamine and eosinophil chemotactic factors from their granules and also form and release prostaglandins, leukotrienes (Brain et al., 1985) and platelet activating factor. Serotonin, a vasodilator in man, does not produce significant change in permeability in rats, however, it appears to be a potent inflammatory agent. (Declerk & Harman, 1983). Neutrophil release LTB 4 , a potent chemotactic agent for stimulating and recruiting more neturophils (Bray et al., 1981) may also release several neutral and acid proteases as well as cationic protein that stimulates the release of the contents of the mast cell granules (Safayhi et al., 1992). The lymphocytes release a variety of peptides called lymphokines that inhibit the migration of macrophages, neutrophils, basophils, and eosinophils, exert cytotoxic actions, induce lymphocyte proliferation or induce vascular permeability. Their peptides include interleukins and interferans (Camp et al., 1986). The kinin system, the complement system and the clotting and fibrinolytic system of plasma provide more mediators than influence inflammation. These mediators enhance the vascular symptoms of inflammation. Stimulation of factor XII or Hageman's factor in the clotting system activates pre-kallikrin to kallikrin, which gets converted into

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kininogen and then to bradykinin, as well as activates more factor XII. The latter converts plasminogen pro activators to plasminogen activator that converts plasminogen to plasmin. This, in turn activates the complement system that mediates a series of peptides and cytotoxic agents.

MEDIATORS OF INFLAMMATION It is recognised that inflammation differs from species to species. In same species from one tissue to another and also in the same tissue according to the type of traumas. Yet the early inflammatory events induced by various trauma in different species and tissues have much in common, as far as sign and symptoms are concerned (Ferreira & Vane, 1979). Several highly active substances are liberated locally in tissue during inflammatory reactions. In different types of inflammation, some mediators may have more prominent role than others. The sequence of mediator release may also be important. For instance, in anaphylactic shock there is an explosive and simultaneous release of histamine, SRS-A, PGE 2 and PGF 2<1 and rabbit aorta contracting substance (Piper & Vane, 1971). However, in the inflammatory response to subcutaneous injection of carrageenan in rat, there is sequential release of the mediators (Willis, 1969) at first; there is an output of histamine followed by the detection of bradykinin. Mter 3 h little PG activity «5 ng/mL) was observed but this concentration gradually rose to an average plateau of 80 ng/mL between 18 h to 24 h.

Once the initial reaction to injury has developed, the subsequent changes within the damaged area depend upon the severity, nature and duration of action of the injurious agents. If this is of brief duration or is rapidly and successfully overcome by the defense mechanism of the host, the inflammatory changes will either resolve completely or subside leaving a variable amount of scar tissue with in the injured area. Some types of persistent stimuli lead to massive continued cell migration and to suppuration (pus formation). Cells and tissue changes caused by mediating substances that originate from various cells or plasma are the salient features of the inflammatory process. Stimuli, which trigger the inflammatory process, activate these mediators or lead to their release. Different groups of cells contain several potent mediators. The cells most studied in this respect are neutrophills (polymorphonuclear neutrophills leukocytes), basophills, mast cells and platelets and to a lesser extent macrophages and lymphocytes. These mediators are released in a sequential manner from the inflammatory site by the effected organelles in which histamine is the first to come followed by serotonin bradikinin, members of prostaglandin series and the other related mediators. The onset of inflammation begins with the migration of various inflammatory cells at the site of inflammation. Once inflammatory cells arrive at the site of inflammation, they release mediators, which control

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the later accumulation and activation of other cells. However, inflammatory reactions initiated by the immune system, the ultimate control are exerted by the antigen itself, in the same way as it controls the immune response. For this reason, the cellular accumulation at the site of chronic infection, or in autoimmune reactions, is quite different from that at sites where the antigenic stimulus is rapidly cleared. Inflammatory mediators are soluble, diffuseable molecules that act locally at the site of tissue damage and infection and more distant sites. These mediators are a) Exogenous mediators b) Endogenous mediators

Exogenous Mediators The bacterial products and toxins can act as exogenous mediator of inflammation. Notable among these is endotoxin or LPS of gram negative bacteria. The immune system of higher animals has probably evolved in a variable sea of endotoxin, so it is not surprising that this substance evokes powerful responses. For example, endotoxin can trigger complement activation, resulting in the form of anaphylatoxins C3a and C5a, which cause vasodilation and increase vascular permeability. Endotoxin also activates "Hageman factor", leading to activation of both the coagulation and fibrinolytic pathways as well as the kinin system. In addition, endotoxin elicits T cell proliferation as have been described as superantigen for T cells.

Endogenous Mediators These mediators are produced from within the immune system itself, as well as other systems. For example they can be derived from molecules that are normally present in the plasma in an inactive form. Mediators of inflammation are also released at the site of injury by a number of cell types. These cell types either contain them as preformed molecules within storage granules, e.g., histamine or rapidly switch on the machinery to synthesize the mediators when required e.g., metabolites of aracidonic acid such as prostaglandins. Further more the inflammatory mediators are early phase mediators and the late phase mediators. The early phase mediators are produced by the mast cells and platelets and are especially important in acute inflammation which includes mainly histamine the first to come at the site of inflammation followed by serotonin and other vasodilators like bradykinin. The chemoattractants like C5a and cytokines such as interleukine-1 (IL-1), IL-6 and tumor nacrotic factor-a are also early phase mediators. Late phase mediators are responsible for the regulation of vascular event occuring later after 6 to 12 h ofthe initiation of inflammation these include the products of arachidonic acid.

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Histamine Histamine is a multifunctional substance exerting influence on many types of cells and physiological processes through a distinct set of G proteincoupled receptors. Generally, histamine modulates inflammation and allergic responses via Hl receptors (Ash & Schild, 1966), gastric acid secretions through H2 receptors (Black et ai., 1972), and neurotransmitter release in the central nervous system via H3 receptors (Arrang et ai., 1983). All of these histamine receptor types are members of the supper family of G protein coupled receptors (Lovenberg et ai., 1999). Recently cloning and characterization of a new class of histamine receptor, H4 has been reported (Nakamura et ai., 2000; Liu et ai., 2001). These receptors clearly documented the expression of H4 receptor mRNA on variety of cell types including peripheral blood mononuclear cells, neutrophils, eosinophils, mast cells and resting CD4+ cells. These findings are highly suggestive of a role for the H4 receptor in immune and lor inflammatory modulation. Histamine is the most important vasoactive inflammatory mediator released at the site of inflammation by the inflammatory cells. It is one of the primary mediators released and plays an important and pivotal role in the development and further progression of inflammatory reaction. However, the contents of histamine dwindle within the first sixty min and antihistaminics have no effect on the delayed permeability response. Thus, histamine is mainly important in the early phase of inflammatory responses and in immediate IgE mediated hypersensitivity reaction. Being a biologically potent low molecular weight endogenous biogenic amine, it is distributed in mast cells and basophil granules. It is also present in human platelets. The release of histamine from platelets (platelet release reaction) is stimulated when platelets aggregate after contact with collagen, thrombin, ADP and antigen- antibody complexes. Platelet aggregation and release are also stimulated by platelet activating factor derived from mast cells during IgE-mediated reaction. It is stored in mast cells and basophils largely complexed to mucopolysaccharide such as heparin. It is synthesized from L-histidine by histidine decarboxylase. Two forms of histamine have been described viz., a storage or bound form in the mast cells and intrinsic or free form. Its wide distribution would appear to make it ideally suited as a mediator of acute inflammation and of acute anaphylactic reaction. Widely distributed in the inflammatory cells, histamine has diverse functions including primary, local dilation of small vessels, widespread arteriolar dilation, local increased vascular permeability by contracting endothelial cells, the contraction of non vascular smooth muscles, chemotaxis for eosinophils and blocking T-lymphocyte function. It acts mainly on the microcirculation through Hl receptors (Buss, 1979). The H2 receptors mediate a number of antiinflammatory effects, including the inhibition of eosinophil chemotaxis but cause the vasodilation.

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It appears, therefore, that the role of histamine in the inflammatory response is transient and other mediators and mechanisms are required to explain the continued vascular and cellular changes characterizing the progression and regression of the overall response.

Serotonin Serotonin (5-hydroxytryptamine) is a biogenic amine that was first discovered in the gut (Vialli & Erspamer, 1933). Serotonin was later shown to correspond to a vasotonic substance found in the serum and was therefore called serotonin (Rapport et ai., 1948) Serotonin is also found in brain (Twarog & Page, 1953) and is involved in a wide range behaviors such as sleep, appetite, inflammation and pain perception, locomotion, thermoregulation and sexual activity (Wilkinson & Dourish, 1991). Furthermore, serotonin drugs are used in the treatment of a number of pathological states such as migraine, depression, and anxiety (Sleight et ai., 1991). The multiple action of serotonin is mediated by the specific interaction of this amine with several receptors. Pharmacological and physiological studies identified distinct receptors that were designated 5HT-1A, 1B, 1e, 1D, 2,3, and 4 (Saudou & Hen, 1994). However, some pharmacological studies suggested the existence of additional serotonin receptor subtypes. Further, the classification of 5HT receptor subtypes takes into account not only their pharmacological profile and coupling with second messenger but also their sequences (Saudou & Hen, 1994). Except for 5HT-3 receptors, which are ligand-gated ion channel receptors, all other 5HT receptors belonging to the large family of receptors that interact with G protein. Serotonin is the most important vasoactive mediator that is stored in the mast cell and basophil granules in rodents. It is an endogenous biogenic amine, which, like histamine, has potent effect on small blood vessels and smooth muscles of certain mammalian species. The possible role of serotonin in acute inflammation has not yet been clearly described. In many animal species, mast cells and platelets contain considerably large amount of serotonin which is released along with histamine by many of the inflammatory agents viz., dextran, turpentine and egg albumin (Singh, 1983). Serotonin increases vascular permeability when injected subcutaneouly in small amounts. Furthermore, in some of the studies it has been observed that animals depleted with serotonin showed poor response to local inflammatory reaction produced by different oedemogenic agents. Serotonin is also capable of dilating capillaries producing contraction of nonvascular smooth muscles. Most of it is stored in the gastrointestinal tract and central nervous system but a large amount is stored in the dense granules of platelets.

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Bradykinin Kinins are produced from precursor kininogen via the action of proteolytic enzymes. Two major biochemical cascades for the production of kinins have been described. One within the cardiovascular system activated in association with the blood clotting and another in many other tissue systems activated following tissue injury and inflammation. Bradykinin is the major kinin liberated in the plasma, while in tissues kallidin (Lysyl-bradykinin) is the major kinin formed. Both bradykinin and lysyl-bradykinin are rapidly degraded by kininases to generate the active metabolite des-Arg9 bradykinin or des-ArglO kallidin, respectively, which also contribute to inflammation and hyperalgesia (Dray & Perkins, 1993). Bradykinin has been more extensively characterized than kallidin. It exerts several proinflammatory effects, including (a) plasma extravasation; (b) stimulation offree radical, prostaglandin, and cytokine production from a variety of cells; (c) stimulation of postganglionic sympathetic neurons to affect blood vessels and to release prostanoids; (d) degranulation of mast cells to release histamine and other proinflammatory mediators; (e) chemotaxis of lymphocytes to the sites of injury; and (D mitogenic activity, possibly relating to tissue repair (Dray & Urban, 1996). In addition bradykinin is one of the most potent endogenous algogenic substance which induces pain by stimulating nociceptors directly (skin, joint, muscle) and by sensitizing them to other stimuli. Indeed there is a strong synergism between the actions of bradykinin and other algogenic substances such as PGs and 5-hydroxytryptamine. The effects of kinins are mediated via two main classes of G proteincoupled receptors, B1 and B2. Additional evidences for B3 receptor have been provided by antagonistic studies through pharmacological and molecular techniques (Farmer, 1995). In humans, B1 and B2 receptors are composed of 353 and 364 amino acids respectively representing distinct gene products. Thus couple primarily through the Gq/ll family ofGTP-binding proteins (Hall, 1997). The B2 receptor is expressed more commonly in many tissues and is the preferred site of action for bradykinin and kallidin. Studies with selective antagonists have shown that B2 receptors make a major contribution to inflammatory pain and hyperalgesia (Sterauka et al., 1988; Heapy et al., 1993; Perkins et al., 1993). On the other hand, B1 receptor is encountered less frequently but can be constitutively expressed in certain tissues such as blood vessels. This receptor is preferentially activated by the metabolites des-Arg 9 bradykinin or des-Arg lO kallidin. B1 receptor expression markedly increases during inflammation or infection. The significance of this behavior is not entirely clear but under these circumstances, B1 receptor makes an important contribution to the hyperalgesia (Dray & Perkins, 1993).

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Several potent B2 antagonists with analgesic and antiinflammatory activity have been made. These include the peptide analogues viz., NPC567, Hoe 140, NPC16731, the dimeric peptide CP-0127 (bradycor) and a non peptide antagonist, WIN 64338 (Hock et al., 1991; Cheronis et al., 1992; Sawutz et al., 1994). Less is known about the B1 receptor, but it has little homogeneity with the B2 receptor (Mauke et al., 1994), and in keeping with current pharmacological studies, antagonists for this receptor will likely be different from those developed at B2 sites. However, both B1 and B2 receptor antagonists are likely to be useful analgesic and antiinflammatory agents. The kinins, bradykinin and lysylbradykinin, are important mediators of inflammatory response. In asthma also they mediate inflammation with potent bronchoconstrictor actions. The endogenous release of nitric oxide may inhibit bradykinin-induced bronchoconstriction (Kharitonov et al., 1999). They are liberated from precursor molecules, kininogens, by the action of various proteases, collectively known as kininogenases. Three types ofkininogens have been identified viz., high- and low molecular weight kininogen and T-kininogen. These molecules are synthesized by the hepatocytes and are released into the plasma, where in addition to releasing kinins, they function as cofactor in the cogulation pathway, inhibitors of cysteine protease enzymes and part of the acute phase response. Three different pathways may lead to kinin formation during inflammation i.e., the generation of bradykinin as a result of activation of the Hageman factor and the production of plasma kallikrein, the production of lysylbradykinin by tissue kallikreins and the action of cellular proteases in kinin formation. As bradykinin is such a potent peptide, its activity and its formation must be carefully controlled. Activation of the pathway is controlled internally by the presence of inhibitors for each ofthe active components. Complement component C1 inhibitor controls the activity of the activated Hageman factor, while a2- macroglobulin and C1 inhibitor act as kallikrein inhibitor. There are a variety of enzymes in plasma that control bradykinin activity, including carboxypeptidase N, which removes the C-terminal arginine residue, thus inactivating the peptide.

Prostaglandins Prostaglandins belong to a family of chemical messengers, which have been studied extensively over the last 20 to 25 years. Prostaglandins were first identified in semen, and received their name because at that time they were thought to origin from the prostate gland. However, it is now well known that the prostaglandins in semen are synthesized mainly by the seminal vesicles, and the prostate makes a small contribution. Further, it has been discovered that prostaglandins are produced in almost all tissues of the body, and are ubiquitous messenger molecules with a variety of actions. They are fatty acids synthesized from arachidonic acid, a long chain

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polyunsaturated fatty acids, and have been classified into groups from prostaglandin A to prostaglandin I according to structural variation in the five-carbon ring at one end of the molecule. There is further subdivision based on the number of double bonds in the two side chains. Prostaglandins are key mediators of inflammation and play critical physiological role in tissue homeostasis and function. Tissue injury and certain other stimuli such as inflammatory stimuli cause the local release of chemical mediators such as prostaglandins (Bolten, 1998). During an inflammatory reaction, there is a rapid activation and recruitment of inflammatory cells such as macrophages and neutrophils, release of numerous cytokines and enzymes, and production of pro-inflammatory mediators. Prostaglandins are synthesized from arachidonic acid, the initial step of which is catalyzed by the enzyme cyclooxygenase. They are implicated in the inflammatory response and in sensitizing nociceptors to the actions of other mediators. Occuring during the acute and chronic inflammatory illness, prostaglandins are produced at the site of inflammation where it is believed that they mediate many ofthe symptoms of inflammation, such as oedema and pain. Arachidonic acid, precursor of prostaglandins, is released from cell membranes by phospholipases. Cyclooxygenase catalyzes the addition of molecular oxygen to arachidonic acid to form initially the endoperoxide intermediate prostaglandin G2 (Peskar & Maricic, 1998). The same enzyme also possesses peroxidase activity that catalyzes the reduction of this prostaglandin to form PGH 2 • PGH 2 may then react with a number of enzymes sometimes called isomerase to become one of the prostagladins or thromboxanes. The prostagladins are characterized by a five-membered ring which determines the type of prostaglandin. Two products of this reaction are PGD 2 and thromboxane A2. Two other prostaglandins are produced, viz., PGE 2 and PGI 2 , which act as mediators of the vascular phases of inflammation. They are both potent vasodilators. In addition, they act synergistically with certain of the vasoactive mediators, such as histamine and kinnis, to increase vascular permeability. They also stimulate osteoclastic bone resorption suggesting that bone erosion in chronic inflammatory disease may be mediated at least in part by prostaglandins. Prostaglandins are found throughout the body in almost all tissues. The physiological actions of prostaglandins are medited through an interaction with specific receptors coupled to different secondary messenger system that ultimately produce an array of biologic effects. The same pathway used to generate prostaglandins for the inflammatory process is used to produce prostagladins in other tissues. Generally, the prostaglandin produced in a tissue is a function of specific cell type. For example, endothelial cells produce primarily the vasodilatory PGI 2 , platelets generate

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the vasoconstrictor ~, renal prostaglandins are involved in regulation of renal blood flow and prostaglandins in the gastric mucosa have cytoprotective or, more appropriately termed, gastroprotective functions. The major difference in the production of prostaglandins is the specific type of cyclooxygenase used in the pathway. Until recently, it was believed that there is only one type of cyclooxygenase throughout the body. However, Simons and colleagues (1989) identified a second form of cyclooxygenase, COX-2 distinguishing it from the originally identified enzyme (Bjorkman, 1998b). Subsequent work has shown that the two enzymes have a similar size, nearly identical enzyme kinetic, and have 75% amino acid homology, with complete preservation of the catalytic sites for cyclooxygenase and peroxidase activity. The genes for the two enzymes are found on different chromosomes. The two enzymes are known as cyclooxygenase-l (COX-I) and cyclooxygenase-2 (COX-2). Under normal homeostatic conditions, COX-l is a constitutive enzyme -present in most cells and tissues-where it functions to produce physiologically important prostaglandins. COX-l and COX-2 enzymes differ significantly both in protein structure and mRNA. The human cDNA for COX-2 has been cloned and expressed (Hla & Neilson, 1992). It encodes a 70 kD polypeptide of 604 amino acids that is 61% identical to human COX-I. The mRNA of COX-2 consists of 4.1 kilobases and is considerabely larger than the 2.8 kilobase mRNA of COX-I. This is consistent with its inducible nature. Cells use COX-l to produce prostaglandins in the continuous response to stimulation by circulating hormones and other factors (DeWitt, 1995). This differs significantly from the role of COX-2 and enzyme that is rapidly induced at the site of inflammation and is responsible for the production of pro-inflammatory prostaglandins (Masferrer et al., 1996; Peskar & Maricic, 1998). Under normal conditions, resting neutrophils, macrophages, endothelial cells, and fibroblasts show little or no COX activity but in response to an inflammatory stimulus there is increased activity due to synthesis of COX-2 (Richardson & Emcry, 1995) distribution studies indicate the presence of COX-l in nearly all normal issue including kidney, spleen, stomach, liver, heart and brain. In contrast virtually no expression of COX-2 in normal tissues is detectable. COX-2 expression is readily induced in response to proinflammatory stimuli in cells in vitro and in vivo, including macrophages, monocytes, synovial cells and endothelial cells. COX-2 is not expressed to a significant degree in resting tissue, except in brain, in reproductive organs and kidneys (DeWitt, 1995). The function or activity of COX-2 in these tissues is not known yet. In the gastrointestinal tract, prostaglandins PGE-2 and PGI-2 are derived from COX-l activity and have been correlated to the long term protection of mucosal lining from damage (Frolich, 1997). COX-l is found in the blood platelets where it is essential for the synthesis ofthrombaxane

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A-2. COX-1 activation leads to the production of prostacyclin which is ant-ithrombogenic when released from the endothelium and cytoprotective when released from the gastric mucosa. This cytoprotection of the gastric mucosa extends to both exogenous damaging substances as well as endogenously produced gastric juice. In the kidney, prostaglandins are synthesized mainly in the renal medula, the ascending loop of Henley, and the cortex. Renal prostaglandins influence several functions including, total renal blood flow, distribution of renal blood flow, sodium and water reabsorbtion and renin released. These functions with the possible exception of renin secretion seem to depend on COX-1 activity. Renin secretion has been linked to COX-2 activity because the presence of COX-2 has been demonstrated in the macula dens a and found to up-regulate with sodium deprivation. A close link between the inflammatory response and COX-2 induction is becoming evident (Frolich, 1997). On the basis of studies using proinfalmmatory and anti-inflammatory cytokines to induce inflammation, COX2 is induced in macrophages, while the anti-infalmmatory cytokine, IL-10 down-regulates COX-2 expression. Synovia obtained from patients suffering from osteoartritis and rheumatoid arthritis shows COX-2 in mononuclear cells, endothelial cells of blood vessels and synovial fibroblast cells. The evidence for the involvement of prostaglandins in arthritic diseases is extensive (Williams & Higgs, 1988; Goodwin, 1991; Malmberg & Yaksh, 1992). The classical inflammation of rheumatoid arthritis consists of redness, oedema, pain and heat. Proataglandins are found at the sites of inflammation and prostaglandins 1-2, E1, E2 and D2 cause vasodilation leading to redness (Robinson, 1991). Although prostaglandins 1-2, E2, and F2 are only weakly active in producing oedema, they contribute synergestically to the tissue swelling induced by other inflammatory mediators such as bradykinin and histamine. The role of prostaglandins in fever is complex and involves interlukin-1 (Rothwell, 1992). The discovery by Vane in 1971 that NSAIDs such as aspirin exert their antiinflammatory activity by inhibiting the COX enzyme has found wide acceptance although part of the mechanism of action of these compounds probably does not involve prostaglandins (Abramson & Wissmann, 1989; Brooks & Day, 1991). The effect of prostaglandins on the immune system is complex, incompletely understood and often controversial. Among the many actions of prostaglandins on the immune system, inhibition of Band T cell proliferation (Bray, 1987), inhibition of rheumatoid factor (Alvarellos et al., 1988) and suppression of antigen presentation by macrophages, probably are the most relevant effects as far as arthritic diseases are concerned. Although it is generally accepted that prostaglandin E2 is largely immunosuppressive by virtue of its downregulation ofB and T cell functions. It has been recently found that this compound also potentiates Ig class switching both indirectly by inhibiting interferon secretion from TH1 cells

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and directly by enhancing IL-4 induced class switching to IgG and IgE (Phipps et al., 1991). T cells, in particular CD4+ helper cells, playa central role in orchestrating the immune responses that underlie rheumatoid inflammation (Cush & Kavanaugh, 1995). Of the various subsets of helper T cells, the Th1 subset in particular is relevant (Miossec & van den Berg, 1997). Growing attention has been paid to the balance of Th1JTh2 subsets of helper cells. The Th1 subset of cells which functions primarily in cell-mediated immune reaction, seems to play the dominant role in rheumatoid arthritis (Miossec & van den Berg, 1997). Therefore, alteration of the Thlfrh2 balance has been suggested to be a practically valuable therapeutic approach. Prostaglandin E2 has been implicated in the angiogenesis required for the spread of the pannus in the synovial hyperplasia component of rheumatoid arthritis (Colville-Nash & Scott, 1992).

Cytokines During the course of non-specific as well as immunologically mediated inflammatory reactions, many hormone-like mediators called cytokines are produced. They regulate local as well as systemic inflammatory host diffense. Unlike many hormones, cytokines are not normally present in plasma and nor are produced by glands, but are autocrine and paracrine mediators that are produced by inflammatory cells and many other tissues in response to invasive organisms, irritants or trauma. Although monocytes and lymphocytes are the major source of cytokines among blood cells yet evident that cytokines are generated by many other cell types (Cassatella, 1995 & 1999). The ability of polymorphonuclear leukocytes (PMNs) to produce such a variety of cytokines readily suggests that these cells might not only be inflammatory and immune responsives, but also other processes such as hematopoiesis, wound healing, angeogenesis, and antiviral defense. PMNs can express proinflammatory cytokines such as TNFa, (Djeu et al., 1990), IL-1a and IL-1P (Lord et al., 1991) and IL-12 (Cassone et al., 1997), antiinflammatory cytokines such as IL-1ra (Fava et al., 1991). Many cytokines have been identified those contribute either directly or indirectly to inflammatory processes. Cytokines along with some growth factors produce specific protein at the site of inflammation (Kujubu et al., 1991). In general the cytokines are soluble (glyco) proteins, nonimmunoglobulin in nature, released by living cells of the host, which act nonenzymetically in picomolar to nanomolar concentrations through specific receptors to regulate host cell function. They make up the fourth major class of soluble intercellular signaling molecules, alongside of neurotransmitters, endocrine hormones, and autacoids. They possess typical hormonal activities i.e., they are secreted by a single cell type, react specifically with other cell types (target cells) and regulate specific vital functions that are controlled by feedback mechanism. They generally act at short range in a paracrine or autocrine (rather than endocrine) manner.

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They interact first with high affinity cell surface receptors (distinct for each type or even sub type) and then regulate the transcription of a number of cellular genes by little understood second signals. This altered transcription, which can be an enhancement or inhibtion, results in changes in the cell behavior. Target cells, on which cytokines transform their information signal, may be localised in any body compartment (sometimes a long distance from the sight of secretion). Other type ofthese molecules mostly on neighbouring cell in the microenviornment where they have been released. These are characterised as local hormones and their secretion is brought about by autocrine or paracrine mechanisms. During the paracrine secretion some cytokines may escape cell binding and may spill over into general circulation via lymph or plasma. This is important, especially for the products of lymphoid cells which are mobile after having picked up the message in the microenviornment through out the body. Therefore, their immunoregulatory products, (Lymphokines, monokines, interleukins and other cytokines), despite being of local hormone character, may infact act systemically. Cytokines are synthesised, stored and transported by various cell types (Cassatella, 1995) not only inside the immune system (lymphokines, monokines, tumour necrotic factor, and interferons) but also by other which are mainly studied by haematology (colony stimulating factors), oncology (transforming growth factors), and cell biology (peptide growth factors, heat shock and other stress protiens). The central role of cytokines is to control the direction, amplitude, and duration of immune responses and to control the modelling and remodelling of the tissues, be it developmentally programmed, constitutive or unscheduled. Unscheduled remodelling accompanies inflammation, infection, wounding and repair. Individual cytokines can have pleiotropic, overlapping and sometimes contradictory functions depending on their concentration, the cell type they are acting on, and the presence of other cytokines and mediators thus the information which an individual cytokine conveys depend on the pattern of regulators to which a cell is exposed, and not on one single cytokine. It is supported that all cytokines form the specific system or net work of communication signals between cells of the immune system, and between the immune system and other organs. In this inter cell signalling network, the signal is usually transferred by means of specific set of cytokines. Because of the potential and profound biological effects of cytokines, it is not surprising that their activities are tightly regulated, most notably at the level of secretion and receptor expression. Additional regulatory mechanisms are provided by the concomitant action of different cytokines and the presence in the biological fluids of specific inhibitory proteins, soluble cytokine binding factors and specific autoantibodies. The cytokine is a very potent force in homeostasis when activation of the net work is local and cytokines act vicinally in surface-bound or diffusible form, but

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when cytokine production is sustained/or systemic, there is no doubt that they contribute to the signs, symptoms and pathology of inflammation, infections, autoimmune, and malignant diseases. TNFa is an excellent example of such duel action. Locally it has important regulatory and anti tumour activity but when it circulates in higher concentrations beyond the organ of origin, it may be involved in the pathogenesis of endotoxic shock, cachexia and other serious diseases. From the inflammatory point of view there are two main groups of cytokines viz., pro-inflammatory and antiinflammatory. Pro-inflammatory cytokines are produced predominantly by activated macrophages and are involved in the up-regulation of inflammatory reaction (Beauerle & Baltimore, 1996) whereas, antiinflammatory cytokines belong to the T cellderived cytokines and are involved in the down-regulation of inflammatory reactions. The cytokines which play the central role in inflammation are IL-1 and TNFa (Arend & Dayer 1990), because the administration oftheir antagonists, such as IL-Ira (IL-1 receptor antagonist), soluble fragment of IL-1 receptor, or monoclonal antibodies of TN Fa and soluble TNF receptor, all blocks various acute and chronic responses in animal models of inflammatory diseases (Dawson et ai., 1991). Some of these antagonists are beginning to utilize as antiinflammatory agents in diseases such as sepsis and rheumatoid arthritis. IL-1 and TNFa together with IL-6 serve as endogenous pyrogens. The up-regulation of inflammatory reaction is also performed by IL-ll, IFNP, IFNy, and especially by the members of cytokines superfamily. On the other hand, antiinflammatory cytokines (IL4, IL-10 and IL-13) are responsible for the down-regulation of inflammatory response. They are able to suppress the production of pro-inflammatory cytokines. Their strong antiinflammatory activity suggests possible utilization in management of many antiinflammatory diseases, including sepsis, rheumatoid arthritis, inflammatory bowl disease, psoriasis, T cellmediated autoimmune diseases such as type-I diabeties, as well as in acute graft-versus-host disease. IL-10 is capable of effectively protecting mice from endotoxin-induced shock, a lethal inflammatory reaction mediated by TNFa and IL-I (Firstein & Zvaifler, 1997). The production of most of the lymphokines and monokines such as IL-I, IL-6 and TNFa is also inhibited by transforming growth factor (TGFa), but on the other hand, TGFa has a number of pro-inflammatory activities including chemoattractant effects on neutrophils, T lymphocytes, and unactivated monocytes. TGFa has been demonstrated to have in vivo immunosuppressive and antiinflammatory effects as well as proinflammatory and selected immunostimulatory activities. When administered systemically, TGFa acts as an inhibitor, but if given locally can promote inflammation. Generally, TGFa stimulates neurovasculariztation and the proliferation and activities of connective tissue cells and is a pivotal factor in scar formation and wound healing. But TGFa has anti proliferative effect on most other cell types including

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epithelial cells, endothelial cells smooth muscle cells, fetal hepatocytes and myeloid, erythroid, and lymphoid cells. TGFa is a potent immunosuppressive cytokine that suppresses cell mediated as well as humoral immunity including tumor immunity.

Products of the Complement System Complement is a complex system containing more than thirty various glycoproteins present in serum in the form of components, factors, or other regulators and/or on the surface of different cells in the form of receptors. These are present in blood serum in an inactive form and are activated by immune complexes (the classical pathway), by carbohydrates (the lectin pathway), or by other substances, mainly of bacterial origin (the alternate pathway). The components of the classical pathway are numbered 1 to 9 and prefixed by the letter C, e.g., CI, C2 up to C9. CI is composed of three subcomponents CIq, Clr and CIs. The early components of alternate pathway are known as factors and each molecule is named by a letter, e.g., factor B, D, P. The lectin pathway is the same as the classical pathway, only CIq is omitted. All these pathways use in the later stages of activation the same terminal components C5-C9 that form membrane attack complex (MAC). C3 also participates in all pathways. The complement system influences the activity of numerous cells, tissues and physiological mechanisms ofthe body. These effects may involve either the whole complement, or only individual components of fragments. Activation of the complement cascade, with the formation of the effector MAC unit, results in cytotoxic and cytolytic reactions. Target cells for MAC action may be heterologus erythrocytes, nucleated cells, bacteria (gramnegative, susceptible to serum), microscopic fungi, viruses with a surface envelope and viruse-infected cells. The results of the cytotoxic complement reaction may be beneficial for the body (elimination of the infectious agent or damaged cells) or harmful (damage to autologus normal cells by immunopathological reactions). The complement system is a potent mechanism for the initiation and amplification of inflammation. This is mediated through fragments of complement components, which belongs to anaphylatoxins. Anaphylatoxins are proteolytic products of the serine proteases of the complement system C3a, C4a, and C5a. The production of anaphylatoxins follows not only from complete activation, but also from activation of other systems which may directly cleave C3, C4 and C5. Such enzymes include plasmin, kallikrine, tissue and leukocyte lysosomal enzymes, and bacterial proteases. C5a is extremely potent for stimulating neutrophils chemotaxis, adherence, respiratory burst generation and degranulation. C5a also stimulates neutrophils and endothelial cells to express more adhesion molecules. Ligation of the neutrophil C5a receptor is followed by mobilization of the membrane arachidonic acid which is mobilized to prostaglandins and

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leukotrienes including LTB 4 , another potent chemoattractant for neutrophils and monocytes. Following ligation of C5a receptor, IL-l is released. Thus, the local synthesis of C5a at the site of inflammation has powerful pro-inflammatory properties. C3b and C4b fragments act as opsonins and enhance phagocytosis. In addition to inducing phagocytosis, ligation of complement receptor on neutrophils, monocytes and macrophages may also stimulate exocytosis of granules containing powerful proteolytic enzymes and free radical production through the respiratory burst which further act as pro-inflammatory. The activation of complement by immune complexes is normally beneficial. Immune complexes bearing C3b are efficiently removed from tissues and from the circulation by monocytes and other phagocytes. However, there are circumstances in which immune complex production continues at a high level; complement complexes may then prove deleterious. Such complexes may form in tissues, e.g., in glomeruli of patients with autoantibodies to glomerular basement membrane (Goodpasture's syndrome) or at motor end-plates in-patients with autoantibodies to acetylcholine receptors (Myasthenia gravis).

Chemotactic Factors The term chemotaxis refers to the movement of leukocytes (or cells in general), induced by a chemotactic stimulus. Beside chemotaxis, which is the stimulated directional migration, leukocytes also possess two other types of movements viz., random migration which is undirected spontaneous migration and chemokinesis i.e., stimulated unidirectional migration. A chemotactic stimulus is provided by substances that can either attract or repulse the cells. Thus, chemotactic cell movements can be either negative or positive, i.e., the cells may move towards the source of the chemotactic substance (towards an increasing concentration gradient) or in the opposite direction. The positive movement is typical for leukocytes. Substances possessing chemotactic activity are called chemotactic factors (chemotaxins, chemoattractants). Leukocyte chemotaxis is mainly responsible for their mobilization at the inflammatory site. Both exogenous and endogenous chemoattractants participate in this event. Exogenous chemotaxins include bacterial oligopeptides, lectins, denatured proteins, some lipids and lipopolysaccharides. Endogenous chemotaxis are produced by the host organism and are of humoral or cellular type. The humoral endogenous chemotaxins are complement fragment C5a, C5desArg, and Ba, fibrinopeptides, kallikrein and plasminogen activators whereas, the cellular component comprised of LTB 4 , platelet activating factor, and chemotactic cytokines. Interaction between the chemotactic factor and its corresponding receptor triggers a series of coordinated biochemical events, which include changes in the cell transmembrane potential, altered cyclic neucleotide

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levels and ion flow across the cytoplasmic membrane and increased glucose utilization and oxygen consumption. The composition of membrane phospholipids is altered and arachidonic acid, released by phospholipases, is metabolized into a number of biologically active intermediates and products. Within a few min, the leukocyte changes from round to a triangular shape that is oriented along the direction of the chemotactic gradient. Reorganisation of cytoskeletal contractile elements, particularly active microfilaments and microtubular structures, contribute to this shape change. Activation of the contractile cell system not only results in the migration but also in other form of movement such as enhanced adherence, spreading, endocytosis and secretion of lysosomal enzymes resulting in the further stimulation of the inflammatory mediators.

The Acute Phase Reactants Within the spectrum of systemic reaction to inflammation two physiological responses in particular are regarded as being associated with acute inflammation. The first involves the alteration of the temperature set-point in the hypothalamus and the generation of the febrile response. The second involves alteration in metabolism and gene regulation in liver. Three cytokines that are released from the site of injury viz., IL-l, TNFa and IL6 are considered to regulate the febrile response, possibly as a protective mechanism. It is important to consider the acute phase response (and inflammation) as a dynamic homeostatic process that involves all of the major systems of the body, in addition to the immune, cardiovascular and central nervous system. Normally, the acute phase response lasts only a few days; however, in case of chronic recurring inflammation, an aberrant continuation of some aspects the acute phase response may contribute to the underlying tissue damage that accompanies the disease, and may also lead to further complications. The second important aspect of the acute phase response is the radically altered biosynthetic profile of liver. Under normal circumstances, the liver synthesizes a characteristic range of plasma proteins at steady state concentrations. Many of these proteins have important functions and higher plasma levels of these acute phase reactants or acute phase proteins are required during the acute phase response following an inflammatory stimulus. Although most of the acute phase reactants are synthesized by hepatocytes, some are produced by the other cell types, including monocytes, endothelial cells, fibroblasts and adipocytes. Acute phase reactants have a wide range of activity that contribute to host defense i.e., they can directly neutralize inflammatory agents, help to immunize the extent oflocal tissue damage, as well as participate in tissue repair and regeneration. There is a rapid increase in the plasma concentration of many complement cascade components the activation of which ultimately results in the local accumulation of neutrophils, macrophages and plasma proteins. These

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participate in the killing of infectious agents, the clearance of foreign and host cellular debris, and the repair of damaged tissue. Coagulation components, such as fibrinogen, play an essential role in promoting wound healing. Proteinase inhibitors neutralize the lysosomal proteases released following the infiltration of neutrophils and macrophages, thus controlling the activity of the proinflammatory enzyme cascade. The major acute phase reactants in mammals include serum amyloid-A and C-reactive protein or serum amyloid-P component depending on the species. Generally, only one of these proteins is an acute phase reactant in a given mammalian species. In humans normal plasma serum amyloid P component levels remain constant during inflammation but C-reactive proteins can increase upto lOOO-folds depending on the disease and its severity. Serum amyloid-A enhances the binding HDL3 to macrophages during inflammation, concomitant with a decrease in the binding capacity ofHDL3 to hepatocytes. The exquisite responsiveness of C-reactive protein to acute phase stimuli, along with its wide concentration range and ease of measurement, have led to plasma C-reactive levels being used to monitor accurately the severity of inflammation and the efficacy ofthe disease management during an infection. Conversely, some diseases are associated with relatively low plasma levels of C-reactive proteins. C-reactive protein and serum amyloidP component are archetypal examples of plasma proteins that are likely to be beneficial during the transient acute phase response but these have detrimental effects in chronic inflammation. Acute phase reactant synthesis is under control performed by inflammatory mediators from which several cytokines and hormones specifically regulate the transcription of human acute phase reactants these include TNFa, IL-l, IL-6, IL-ll, IFNy, TGFa and glucocorticoids. In addition, insulin and akadaic acid have been shown to act as inhibitor of the cytokine-derived induction of some acute phase reactants. Most of the increase in acute phase reactant biosynthesis is due to increase (or decrease) gene transcription.

Caspases Caspases are a family of intracellular cysteine proteases that clear their specific substrates at aspartic acid residues. These proteases have been implicated in two aspects of cytokine biology viz., proteolytic processing and bioactivation of the proforms of certain interleukins such as pro-IL-l~ and pro lL-18 and signaling by apoptosis inducing members of the tumor necrosis factor receptor family, such as tumor necrosis factor receptor-l (TNFR-l) (CD120a) and Fas (CD95). Abundant evidence, derived from molecular biological, genetic, and pharmacological studies, has demostrated a critical role for specific caspases in the bioactivation or bioactivity of particular cytokines. Moreover, these caspases to play nonredundant roles in a wide variety of cytokine mediated inflammatory and autoimmune

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diseases. The involvement of caspases in cytokine biology is examined, with emphasis on relevance to infectious diseases, inflammation and autoimmunity. Ten members of the caspase family of cysteine proteases in humans have been described (Alnemri, 1997; Salvesen & Dixit, 1997). These proteins are initially synthesized as single polypeptide zymogens that undergo proteolytic processing at specific aspartic acid residues to produce the active enzyme. The active, processed caspases are composed of heterotetramers, containing two large and two small subunits, which form two active sites per molecule. Caspases represent a unique family of intracellular cysteine proteases, with absolute specificity for aspartic acid in the P-1 position of substrates. This fact, together with the observation that procaspase zymogens are processed at aspartic acids to produce the active proteases, creates opportunities both for auto activation of caspases and for cascades of caspase activation in which one active member cleaves and activates another. The N-terminal region of the zymogens is typically but not always removed from the protein during processing. Caspases can be grouped into two broad categories (a) the caspase-1 like family and (b) the caspase-3 like family. The categorization of caspases into subfamilies is based on differences in their substrate preference, comparisons of their primary amino acids sequences, and depending on whether their primary function is most relevant to cytokine processing versus apoptosis regulation. The human caspase-1 subfamily consists of caspase-1, 4 and caspase-5. The murine caspase-II protein is probably the orthologue or a close homologue of human caspase-4 (Wang et az', 199B). These caspases share extensive aminoacid sequence identity and are intimately in proinflammatory cytokine processing. The human caspase-3 subfamily consists of caspase-2, caspase-3, caspase-6, caspase-B caspase-9 and caspase-10. Compared to the caspases 1 subfamily, these caspases generally share greater primary amino acid sequence. The members of the caspase-3 subfamily are initimately involved in apoptosis, functioning either as upstream initiators or down stream effectors of this cell death process. Based mostly on differences in their prodomains, the caspase-3 subfamily can be further divided into three additional groups. Abundant evidence implicates caspases in inflammatory and autoimmune conditions mediated by cytokines. The production of bioactive interleukin" a critical mediator of endotoxin shock, is absolutely dependent on caspases-1 in vivo. Similarly, proteolytic processing of prointerleukin-1B, a major inducer of interferon-y in vivo, appears to be mediated exclusively by caspase-l. Cytolytic T-cells and natural killer cells also induce caspase-1 activation. The central importance of caspase-1 as a mediator of inflammatory responses is further supported by the observation that some types of bacteria produce proteins that directly bind to and activate caspase-1, whereas some viruses express proteins that directly inhibit this protease.

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Two forms of interleukin-1, IL-1a and 1L-1~ bind to the same receptor(s) explaining their essentially superimpossible bioactivity profiles (Dinarello, 1994). Both IL-1a and 1L-1~ are produced initially as intracellular proproteins which require proteolytic processing to result in secretion from cells. These cytokines are principally produced by activated macrophages, though some other cell types can also produce 1L-1~, including microglial cells, endothelial cells, vascular smooth muscle cells and epidermal langerhans cells. 1L-1~ stimulates proinflammatory responses in neutrophils, endothelial cells, synovial cells, osteoclasts and other cell types (Dinarello & Wolff, 1993). 1L-1 has been implicated in a wide variety of inflammatory conditions in vivo. Multiple animal model experiments have demonstrated a critical role of 1L-1 for inflammatory conditions including arthritis, inflammatory bowel disease. Clinical studies in patients with rheumatoid arthritis suggest that soluble 1L-1R or 1L-1RA may reduce joint tenderness and inflammatory symptoms. (Campion et al., 1996; Drevlow et al.,1996). The in vivo importance of caspase-1 for inflammatory diseases has also been confirmed by animal model experiments using peptide inhibitors of caspase-1 also significantly reduced collagen-induced arthritis in mice. Caspase-1 inhibitors are superior to indomethacin and prednisolne in this animal model of arthritis (Ku et al., 1996). Similarly, the caspase-1 antagonist benzyloxy-valingy alanyl-3(s)-3-amino-4-oxo-5-(2,6-dichlorobenzoyl-oxy acid) ethyll ester, which inhibits LPS - induced IL-1~ secretion from a monocyte cell line with an IC 50 of24 mm, suppressed carrageenan-induced paw oedema in rats by upto 60% when administered 1 h after the inflammatory agent (Elford et al., 1995). Though overexpression of caspase-1 can induce apoptosis (Miura et al., 1993), lack of caspase-1 does not interfere with the normal cell turnover required for tissue homeostasis, caspase-1 knockout mice develop normally and have no apparent increased incidence of tumors or other anomalies (Kuida et al., 1995). Moreover, absence of caspase-1 does not interfere with any immune system functions. The T-cell proliferative responses to listeria monocytogenes and delayed type hypersensitivity reactions for example, are reportedly normal in caspase deficient mice, suggesting that the inflammatory cytokines generated by caspase-1 dependent processing may be more relevant to macrophage-mediated inflammatory responses than to T cell-dependent immune reactions (Gu et al., 1997) Tumor necrosis factor-a (TNF-a) has been implicated at some level in most inflammatory and auto immune diseases, as well as in graft rejection, heart failure and other conditions LPS-induced production ofTNFa is some what reduced in caspase-1 and caspase-II knockout mice (Wang et al., 1998) but probably not enough to be of physiological relevance. Interferon-y, which is linked to the pathogenesis of inflammatory and other autoimmune diseases are elevated indirectly by caspase-l. The indirect mechanism of caspase-1 production of interferon-y has been demonstrated by experiments

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in caspase-1 knockout mice (Gu el al., 1997). Hence caspase-1 represents an attractive target for treatment of autoimmune, inflammatory and other diseases when these cytokines are implicated. It is quite evident that caspase are absolutely required for the production of several proinflammatory cytokines including IL-1a, IL-1~, IL-18 and probably interferon-yo These cysteine proteases also playa requisite role in the signaling mechanisms. Human diseases involving these cytokines therefore, are potentially amenable to modulation by caspase inhibitors. Further investigations of the effects of caspase inhibitors are now required. From this analysis, clinical indications for caspase- inhibiting drugs can be deducted and appropriate clinical trials initiated to test their efficacy in humans.

Nitric Oxide Nitric oxide (NO), a small hydrophobic inorganic gas molecule, highly versatile, potent, short lived and free radical which acts as a unique neuronal messenger molecule for both intra and intercellular messages involved in the regulation of diverse physiological processes including smooth muscle contractility, platelet reactivity, central and peripheral neurotransmission and the cytotoxic actions of immune cells. It is a relatively stable unchanged radical that readily crosses lipid membranes and interacts with a few specific targets. It also reacts with other species possessing unpaired electrons, to yield secondary products that are often more reactive. It has become a species of extreme biological interest due to the diversity of its physiological functions and general ubiquity. NO is crucial for many physiological functions, an inappropriate release of this mediator has been linked to a number of pathologies. Thus agents that modulate the activity of NO may be of considerable therapeutic value, in particular that reduce the formation of NO. It may be beneficial in pathophysiological states in which excessive production of NO is the contributory factor. These include diseases, such as septic shock, neurodegenerative diseases and inflammation. In fact NO is a colorless gas at room temperature and pressure. Its maximum solubility in water is similar to that of molecular oxygen, 2-3 nM. It is a fairly nonpolar molecule which could be expected to freely diffuse through membranes. One of the most unique and outstanding chemical features of NO is that it is a paramagnetic species. Using the most basic bonding description, it is immediately evident that NO has an unpaired electron. Although much of the chemistry of NO is dominated by the fact that it is a radical, it does not possess the type of reactivity normally associated with other radicals. For example, unlike other, carbon, oxygen or nitrogen centered radicals, NO does not even have the tendency to dimerize i.e., at standard temperature and pressure, NO tends to remain in monomeric form. This lack of dimerization has been attributed to the fact

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that overall bonding does not increase when two NO molecules interacted (Palmer et al., 1988) thus, the formal bond order in NO is 2.5, while the bond order for ONNO is 5. 0= N-N=O Bond order 2.5

Bond order = 5 (still 2.5 bonds per NO)

NO which is one of the smallest biologically active messenger molecules has a wide range of physiological and pathological actions. It is synthesized from the terminal guanidino nitrogen atom of L-arginine by the action of cytosolic enzyme nitric oxide synthase (Hibbs et al., 1987, Knowles et al., 1989, and Bredt et al., 1991). Nitric oxide synthases (NOS) constitute a family with atleast three distinct isoforms. Inorder of their molecular characterisation these include neuronal (cNOS) (Murphy et al., 1993), inducible (iNOS) (Leone et al., 1991) and endothelial (ecNOS) (Marietta, 1991) synthases. This nomenclature is based on the tissue of origin from which original protein and cDNA were first isolated. Although the three isoforms have different molecular weights and variable cofactor requirements, all of them are dependent on NADPH, and show similarities with cytochrome P-450 reductase and also with the bacterial enzymes sulfite reductase and cytochrome P-450 BM3. The formation of NO by NOS is linked to incorporation of molecular oxygen into the molecule (Bredt & Snyder, 1990). NOS has been proposed to form NO and L-citrallin in two steps, the first step being the formation of N-hydroxy-L arginine and in the second, its three electron oxidation. Both steps may utilize different heme-based oxidants i.e., a perferryl species, [FeOO]+, for the first step and a peroxoiron species (FeO)3+ for the second step. Both of these are produced when heme reacts with molecular oxygen (Moncada et al., 1991). All forms of NOS contain four prosthetic groups, flavin-adenine dinucleotide, flavin mononucleotide, tetrahydrobiopterin and a heme complex, iron protoporphyrin IX. All NOS isoforms are dependent on calmodulin. In the inducible isoform calmodulin is already present in a tightly bounded form. The role of calmodulin is to control the electron transfer from NADPH to the flavins, possibly by causing a reorientation of the reductase and oxygenase domains of NOS, perhaps moving them into a favourable position for electron transfer between them. The function of tetrahydrobiopterin is unclear, but may substitute for peroxy-heme to carry out nucleophilic attack on hydroxyarginine or perhaps may act as a stabilizer of the active structure of NOS (Moncada et al., 1991). cNOS and ecNOS are the constitutive isozymes and are calcium! calmodulin dependent, whereas iNOS is absent from the mammalian cells but under physiological conditions is induced by proinflammatory stimuli,

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such as bacterial lipopolysaccharide or the cytokines viz., tumor necrosis factor (TNF), interleukin-1 (IL-1), or interferon-y UFN-y), as well as their combination. cNOS and ecNOS constitutive isozymes, when activated, release short "Puffs" of NO from neurons and endothelial cells which is important in both intercellular and intracellular signalling (Charles et ai., 1993). iNOS, the inducible isozyme is induced in many cell types including vascular smooth muscle cells, endothielial cells, hepatocytes macrophages, neutrophils, chondrocytes and cynoviocyted (Stadler et ai., 1991; StefanovicRacic et ai., 1993) in response to inflammatory and immunologic simuli. iNOS generates much larger quantities of NO over longer period of time (nanomoles, rather than picomoles). The NO generated by macrophages activated by cytokine and endotoxin contributed to their cytotoxic and cytostatic properties against target cells (Palmer et ai., 1993). Although NO generated by constitutive NOS appears to be beneficial in many physiological processes, the excess of NO generated by iNOS has been implicated in the pathogenesis of various inflammatory and immunologically mediated diseases including graft-vs-host, diabetes, viral infections and arthritis (Ialenti et ai., 1993) During inflammation profound physiological changes are often observed including vasodilation, fever and activation of the immune system. These changes are induced by a myriad of cellular mediators, such as endotoxin, various cytokines and eicosanoids. It has been established that NO plays an important role as one of these mediators in addition to it's described functions in cell-cell communications and neurotransmission. As the product of three isoforms of NO synthases, it is the iNOS which is expressed by a wide variety of cell types in response to stimulation (Nissler & Billiar, 1993) producing much large quantities of NO relative to the two other constitutive isoforms during sepsis and inflammation. Cell type that expresses iNOS in response to stimulation by certain cytokines and microbial products includes hepatocytes, macrophages, Kupffer's cells and chandrocytes. The regulation of iNOS expression is quite complex, as it involves a variety of mechanisms within a wide range of cell type. For example, the factors that have been important in the rodent for regulating iNOS expression within hepatocytes, as compared to nonhepatocytes, include transcriptional, posttranscriptional as well as posttranslational mechanisms. Regulation of iNOS gene expression is helped by calmodulin molecules to be fully activated at basal levels of calcium allowing the transcription of enzyme. Thus, transcription with subsequent translation of the iNOS gene appears to play an important role in its regulation. Transcription of the iNOS gene is controlled, both positively and negatively, by a number of inflammatory mediators present during infection and inflammation such as interferon- y, tumor necrosis factor ex interleukin-1~, interleukin-2 and lipopolysaccharide (Xie et ai., 1992; Feldman et ai., 1993; Lorsbach et ai., 1993; Vodovotz et ai., 1993). The

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expression of iNOS response to these agents varies among different cell types and there is often strong synergy among many of these agents. The specific mechanisms by which transcription of the iNOS gene is initiated appear to be quite complex and remain to be elucidated, however, the possible regulatory sequences within the promoter, regulatory region in both mouse and human iNOS has been identified. iNOS gene expression can also be regulated at posttranscriptional level, possibly by altering the stability of the newly transcribed mRNA. As L-arginine is the sole physiological nitrogen donor for NO formation, it is clear that regulation of its availability could present one method of controlling cellular rates of NO systhesis. Intracellular arginine may be increased via one of three mechanisms, through uptake from the extracellular fluid, by intracellular protein degeneration, or by endogenous synthesis. There is no evidence that the availability of arginine as a substrate for NO formation is affected by change in the rate of cellular protein degradation. Hepatocytes, macrophages, and pulmonary artery endothelial cells have been shown to up-regulate arginine transport in response to inflammatory mediators that stimulate the production of NO (Bogle et al., 1992; Inove et al., 1993; Lind et al., 1993). NADPH is an essential cofactor in NO synthesis, as it functions as an electron donor (Stuehr & Griffith, 1992). One and a half NADPH molecules are required for each molecule of NO production. Correlation between the formation of NO and the activity ofNADPH-generating pentose phosphate pathway, as well as the rate limiting enzyme glucose-6phosphate dehydrogenase has been made (Blachier et al., 1991; Corraliza et al., 1993). These correlations suggest that NADPH may be up-regulated by the same inflammatory mediators that induce NO formation. Tetrahydrobiopterin, another essential cofactor in NO synthesis (Stuehr & Griffith, 1992) is thought to be involved in the binding of two inactive iNOS isomers into the active dimeric form. During sepsis and inflammation a wide array of cell types are induced to express iNOS, which then produces large amounts of NO, that may then interact with various target molecules, such as oxygen, that groups and metals within the prosthetic groups of various enzymes resulting in their activation or inactivation. In addition, NO may interact directly with DNA as well as with toxic oxygen radicals, either potentiating their toxicity or neutralizing them. The cellular response to NO are dependent on the cell types producing NO as well as the local environment, as the effects of NO appear to be primarily limited to paracrine and autocrine activities as a result of the short biological halflife of NO and its interaction with oxyhemoglobin in the blood stream. Thus, it results in the production of inactive nitrate (N0 3-) and methemoglobin. However, there have been suggestions that NO may exert some endocrine like activity by binding to albumin and glutathione, there by forming stable nitrosothiols that may

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circulate and release active NO at distant sites (Gaston et al., 1993; Keaney et al., 1993). There is evidence that NO can potentiate as well as ameliorate the toxicity of circulating oxygen radicals often seen during sepsis and inflammation as well as during the periods of reperfusion. Rubbo et al. (1995) implicated NO involvement in oxygen radical mediated membrane lipid peroxidation. In contrast, in a perfusion liver model exposed to endotoxin, the inhibition of NO synthesis increases superoxide (0- 2 ) mediated injury (Bautista & Spitzer, 1994). It appears that NO can combine with 0- 2 to form the intermediate peroxynitrite, which is then protonated to peroxynitrous acid. Depending on the pH of the local environment, peroxynitrous acid degrades to either inactive metabolites or toxic radicals. Within an alkaline environment peroxynitrous acid adopts a cis configuration, with subsequent decay to the inactive metabolite nitrate (N0 3 ). However, under acidic conditions peroxynitrous acid adopts trans configuration and yields toxic products, hydroxy radical (OH) and nitrogen dioxide (N0 2 ). Thus, it appears that NO may behave either as a toxic free radical or as an oxygen radical scavenger, depending on the local conditions. The roles of constitutive and inducible NOS isoforms in inflammation have been extensively studied (lalenti et al., 1993). It is likely that NO from constitutive eNOS plays a role in the early stages of inflammation as a mechanism to decrease and limit the process by inhibiting white cell activity and platelet aggregation (Moncada et al., 1990) and by inducing vasodilation (Kajekar et al., 1995). In contrast, NO from iNOS contributes too many aspects of chronic inflammation. In models of acute and chronic inflammation, it has been shown that constitutive NOS accounts for majority of NO activity in early parts of the process, at this point, some polymorphonuclear cells and a few resident macrophages express iNOS. At the peak of chronic inflammations, there is an eightfold increase in total NOS activity, of which greater than 90% can be attributed to iNOS in activated macrophages. The NOS activity is substantially reduced after 14 days as the inflammation disappears. The use of selective iNOS inhibitors may be of benefit in management of chronic inflammatory processes. Chondrocytes in culture express iNOS (Charles et al., 1993) and decreases their proteoglycan synthesis when stimulated by inflammatory cytokines. Adjuvant arthritis in rats in exacerbated by L-arginine and reduced by NOS inhibitors (lalenti et al., 1992). In rheumatoid arthritis elevated concentrations of nitrite and nitrate have been found in synovial fluid of patients, which suggests an increased local release of NO (Farrell et al., 1992). However, it is likely that NO plays a role in down-regulating the activity of osteoclasts. Furthermore, NOS inhibitors have been shown to potentate bone resorption and enhance bone loss in animals.

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Psoriasis lesions which is a chronic inflammatory disease, contains high levels of iNOS, which is absent from healthy skin and there is increased expression of iNOS (Bruch et al., 1996). Further inflammatory conditions in which excessive NO production by iNOS has been demonstrated include asthma and inflammatory bowel disease (Allcan & Kubes, 1996). Furthermore, a consequence of the release of early mediators in inflammation is the modulation of threshold and sensitivity of nociceptors leading to inflammatory pain. Prostaglandins play a major role in this process. NO may also be involved but it is not clear at this stage whether NO enhances or decreases the pain threshold. The ability of NO to reduce inflammatory pain may also underlie the anti-nociceptive effects of endogenous compounds such as opiates (Ferreira et al., 1991). Thus, NO may itself possess anti-nociceptive actions. In human studies involving pain in the hand vein, NO also appears to exert nociceptive effects. Following administration of bradykinin or hyperisomolar solution to isolated perfused hand vein, NOS inhibitors markedly reduced the discomfort (Kindgen & Arndet, 1996). As such, this profile of activity suggests a possible therapeutic role for cNOS inhibitors in reducing inflammatory pain.

Free Radicals The role offree radicals have been implicated in causation of several diseases such as liver cirrhosis, atherosclerosis, cancer, diabetes, ageing and inflammatory disorders etc. and the drugs that can scavenge oxygen radical have great potential in ameliorating these disease processes. Although, the body has several mechanisms to protect the action of oxygen radicals but at times these protective mechanisms are not sufficient when compared to the insult caused to the body. Supplementation of non-toxic antioxidants may have a chemoprotective role in the body in these conditions. The superoxide anion (0-), H 2 0 2 and hydroxyl radical (OR-) are the major ROS which induce cell degeneration by increasing lipid peroxidation of cell membrane, break DNA strand and denaturate cellular proteins resulting in further tissue damage. The natural cellular antioxidant enzymes include superoxide dismutase (SOD), which scavenges superoxide radicals by speeding up their dismutation, Catalase (CAT) a haeme enzyme which removes H 20 2 and glutathione peroxidases (GPX), selenium- containing enzyme which scavenges H 2 0 2 and other peroxidases. Detoxification of the superoxide anion is not a terminating step in free radical scavenging, since the enzymes catalysed dis mutation results in the production ofH20 2 which accumulates in the mitochondria and cytosol. Unless the peroxide is scavenged by CAT and GPX, it in the presence of iron, may also lead to production of OR-. These ROS together with singlet molecular oxygen may attack lipids, proteins and DNA of cells following increased lipid peroxidation chain reactions resulting in wide spread cellular injury and ultimately pathophysiological conditions.

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A significant understanding ofthe reactive oxygen intermediates (ROn and reactive nitrogen intermediates (RNI) in inflammatory diseases is underway. During last two decades interest in the role of free radicals in diseases with an inflammatory component, such as rheumatoid arthritis, inflammatory bowel disease, pulmonary emphysema, atherosclerosis, and neurodegenerative disorders, grew rapidly. Most research into the role of free radicals in inflammation was directed toward the putative role of ROI as damaging agent. Many studies centered on the oxidative modification of biomolecules such as 0.1- antitrypsin, cartilage proteoglycans and collagen, low-density lipoprotein, DNA, lipids, immunoglobulin G, and so on (Halliwell & Gutteridge, 1985; Merry et al., 1989). The cytotoxicity of free radicals and their reaction products was also investigated, particularly in relation to enthothelial cell function (Kus et al., 1995) The free radicals were studied in context of a direct, destructive role in biology. In the course of the present decade researchers have increasingly recognized this cellular control aspect of free radical action. Endotheliumderived relaxing factor has been identified as nitric oxide (NO) (Snyder, 1995) and ROI can activate apoptosis, programmed form of cell death, at least under some circumstances (Jacobson, 1996). In these cases, ROI activity plays a critical role in regulating the expression of genes that encode either proteins with potential proinflammatory action, or proteins that may act in a protective fashion such as heat shock protein 32, also known as heme oxygenase (Willis et al., 1996). Fibroblast proliferation and collagen gene expression, associated with the late wound-healing phase of inflammation, have also been linked to intracellular ROI activity (Houglum et al., 1994). Perhaps the key to future antioxidant drug development for the treatment of inflammatory diseases lies in our ability to modulate the previously mentioned ROI-mediated control pathways when they become perturbed as part ofthe inflammatory process. The inappropriate activation of the responses referred to earlier, such as overexpression of the particular genes, excess NO production, and apoptosis, may be involved in disease progression. Nevertheless, it should be remembered that these processes, when properly controlled, are physiologicaL Therefore, ROIIRNI can no longer be regarded solely as damaging species whose complete elimination by antioxidants is bound to have beneficial effects on human health. Indeed, it can be argued that the converse may be the case. In case of treatment of inflammatory diseases, it may be necessary to define the stage of the disease at which a particular antioxidant therapy may most effectively be used, e.g., during acute inflammatory episodes, or in chronic inflammation, or prophylaxis. Another factor that must be taken into account is the individual variation in sensitivity within the population

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(Harris et al., 1994), as well as the different susceptibilities to oxidative stress of different organs (Fraga et al., 1990), tissues (Kristal et al., 1994) and subcellular locations (Richter et al., 1988). An increased susceptibility to the actions of ROI on target lymphocytes of patients with autoimmune disorders could thus be an important mechanism in the pathogenesis of disease such as rheumatoid arthritis by inducing excessive inflammatory responses and increased clonal proliferation of autoreactive lymphocytes via the activation of transcription factors like NF-KB. These considerations pose a challenge in the search for appropriate antioxidant therapies in human inflammatory diseases. It is suggested that the successful therapeutic manipulation of cellular responses by antioxidant drugs will necessitate the maintenance of a critical balance between free radical activity and antioxidant selectively to specific cell types, or even defined subcellular locations, and within a narrow "concentration window" that blocks "inappropriate" cellular responses, but leaves the physiological levels of free radical activity necessary for normal cell function.

Adhesion Molecule Leukocytes are the principal actors in the body's defense system against invading microorganisms (Wissman & Cooper, 1993). This defense system has a nonspecific branch consisting of granulocytes and macrophages and a specific branch of lymphocytes. Granulocytes (neutrophils, eosinophils and basophils) release cytotoxic compounds from their intercellular granules to their local environment when they encounter microorganisms. This random destruction happens rapidly but it may also harm healthy tissue of the body. Macrophages, the other class of defense cells from the nonspecific immune system, can ingest and destroy microorganisms by phagocytosis or, in a similar way to granulocytes by the secretion of cytotoxic compounds. However, macrophages can also act more specifically by collaborating with lymphocytes and their products. The lymphoid system comprises the cellular components responsible for antigen specific immune defense. B-Iymphocytes produce antibodies that bind to foreign organisms and facilitate their destruction, either by activating the complement system which in turn can perforate the membrane, or by opsonizing the microorganisms, i.e., trigger phagocytosis due to receptors for antibodies and the macrophage surface. T-Iymphocytes act mainly by cell-to-cell contact. One subpopulation of T-Iymphocytes recognizes and kills cells which bear foreign antigen; the second subpopulation helps activity of other hemopoietic cells in the immune response or helps to multiply effector cells. All of these leukocytes patrol the body by circulating through the blood and lymphatic system ensuring a continuous surveillance which is a prerequisite for efficient defense (Anderson et al., 1982). Upon tissue damage

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and inflammation, leukocytes are recruited from the blood to sites of injury, and this trafficking displays exquisite specificity (Butcher, 1991; Dunon et at., 1993; Springer, 1994). For instance, neutrophils selectively enter sites of acute inflammation or tissue damage, while eosinophils extravasate into sites of allergic reactions and parasitic infestations. The migration of lymphocytes is even more selective and includes a complex pattern of recirculation that relates to differentiation and activation. To efficiently protect the body from infectious organisms, the cells of the immune system circulate as nonadherent cells in the blood and lymph, and migrate as adherent cells into tissues when necessary. Rapid transition between adherent and nonadherent states is the key to the dual function of immune surveillance and responsiveness. Circulating lymphocytes in the blood have first to adhere to and then to cross the endothelial lining in order to enter the various lymphoid tissues which are involved in recirculation. One exception is the spleen where there is unhampered access of blood leukocytes to parenchyma (Fawcell, 1994). Three classes of adhesion molecules are involved in leukocytes endothelial interactions. These are the selectins, integrins, and members of immunoglobulin (Ig like) superfamily.

Selectins The selectins are a family of adhesive receptors found on leukocytes (L), platelets and endothelial cells (P) or endothelial cells alone (E) (McEver, 1994). They are designated as L-selectin (CD 62L), P-selectin (CD-62P) and E-selectin (CD 62E). They mediate the initial, low-affinity adherence of leukocytes to endothelium, manifested by rolling along the endothelial cell surface under conditions of flow. These receptors belong to a family because they share a common mosaic structure consisting of an aminoterminal C-type lecting domain, a single epidermal growth factor like domain several short consensus repeats similar to those found in regulatory proteins that bind complement, a transmembrane domain, and a short C-terminal cyto plasmic domain. Selectins are known for their binding to carbohydrate ligands via the lectin domain (Rosen & Bertozzi, 1994). L-selectin was first identified as a homing receptor for lymphocyte migration to peripheral lymph nodes (Watson & Bradlley, 1998) and was found to mediate the binding of lymphocytes to high endothelial venules in peripheral lymph nodes. It has since been found to be constitutively expressed on most leukocytes, including lymphocytes, monocytes, neutrophils (PMN) and eosinophils. Leukocytes can be induced to shed Lselectin from their surface by a variety of activating agents such as formylpeptide, phorbol ester, and lipopolysaccharide. P-selectin is stored in the Weibel-Palade bodies of endothelial cells and the a granules of platelets (Hartwell et at., 1998). Its expression on endothelial cells can be induced by a variety of stimuli, including thrombin, histamine, and phorbol

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esters. In response to this stimulation, P-selectin is rapidly redistributed to the cell surface, resulting in expression within min. In some endothelial cells P-selectin is also induced by cytokines or LPS. E-selectin is expressed on the surface of endothelial cells only following stimulation. Tumor necrosis factor-a (TNF-a), interleukin-l (lL-l), Substance P, LPS, and several other stimuli have been found to induce the expression of E-selectin (Vestweber & Blank, 1999). Eselectin expression requires de novo protein synthesis with transcription and translation resulting in peak surface expression in 4-6 h following stimulation in vitro with a gradual decrease to basal levels by 24 h.

Integrins The second class of adhesion molecules involved in leukocyte traffic is the integrin family involved in extracellular material/cell and cell to cell interactions (Hynes, 1992). The term integrin refers to the ability of these molecules to integrate the intracellular media with the extracellular environment. Currently, integrins are considered as a wide family of celladhesion receptors, with a broad pattern of cellular expression that exerts a key role as modulator of major cellular events, such as proliferation differentiation and death. Integrins are transmembrane heterodimers consisting of non-covalently linked a and ~ chains. Integrins are subclassified based on the specific ~ sub unit. The sub classes that have been found to be involved in the leukocyte endothelial cell interactions are the ~2 integrins, the ~l-integrin, and the ~7-integrin. The three ~2 integrins are leukocyte restricted and are composed of a common ~2 sub-unit (CD18) that is noncovalently linked to one of three a subunits. Cell surface expression of ~2 integrins is not sufficient for adhesion of leukocytes to endothelial cells. Activation of the ~2-integrins due to a change in conformation or postreceptor events is required before adhesion occurs (Aplin et al., 1998). This activation is induced by soluble agents like cytokines, chemotactic factors and coagulation factors etc. and by ligation of other cell surface receptors. The ~1 integrin consists of a common ~1 subunit (CD 29) noncovalently linked to a a subunit and are found on nearly all cell types. However, one ~l-integrin, VLA-4, is found primarily on leukocytes (Werr et al., 1998). It is constitutively expressed on all circulating leukocytes except PMN and like ~2 integrin requires activation in order to bind. The ~7 integrin is expressed on some Band T lymphocytes and functions as the homing receptor for intestinal lymphoid tissue (Carlos & Harlan, 1994). Immunoglobulin Superfamily Members of the immunoglobulin superfamily constitute the third class of adhesion molecules. The superfamily consists of cell surface proteins that

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are involved in antigen recognition and complement binding, as well as cell adhesion. Endothelial members of this superfamily involved in leukocyte endothelial cell adhesion.

Adhesion Cascade and Inflammation The sequence of events involved in leukocyte adherence to vascular endothelium and transmigration into tissue is complex and has been termed the adhesion cascade (Springer, 1994). The first step involves a random contact of the leukocytes with the endothelial cell surface. After initial contact, relatively weak binding of L, P, and/or E selectin with their carbohydrate ligands occurs. Under conditions of flow, these multiple weak interactions are sufficient to slow the leukocyte down, and the leukocyte is seen to roll along the surface of the endothelial cells. The next involves further activation of the endothelial cells and leukocytes by cytokines, chemoattractants, and chemokines that are produced locally (Berlin et al., 1995). This leads to the upregulation and/or activation of the leukocyte integrins and the endothelial Ig-like molecules. The interaction of these two classes of adhesion molecules mediates the firm adherence ofleukocytes to the endothelial cell surface (Huber et al., 1991). The firmly adherent leukocytes migrate across the endothelial cell surface via integrin-Ig like interactions, diapedesis between endothelial cells and then migrate to the sub endothelial matrix via integrin binding to matrix components. (Feng et al., 1998). While the adherence cascade functions normally to recruit leukocytes to extravascular sites for host defense and repair, under some circumstances leukocytes are firmly adhere to the endothelium, a protected microenvironment is found. In this microenvironment, inflammatory mediators produced by the leukocytes may potentially reach high concentrations and overcome local and systemic antiinflammatory protective mechanisms, thereby allowing endothelial cell injury to occur resulting in an increase in microvascular permeability and hemorrhage. This series of events may initiate and sustain a cycle of inflammation, leading to further leukocyte recruitment and endothelial cell injury. The continued recruitment of leukocytes can lead to occlusion of the microvasculature by leukocyte aggregates, causing local ischemia. The leukocytes may also diapede between endothelial cells, gaining access to the extravascular space, where they can mediate further tissue damage, producing organ dysfunctions. This cascade of events leading to vascular and tissue injury dependent on leukocyte-endothelial adhesive interactions, and thus, interrupting the interactions at the level of rolling, firm adherence, or diapedesis should decrease leukocyte mediated injury, in other words inflammation. In addition to inflammation, integrins and selectins are also involved in other abnormal conditions, such as septic shock, thrombosis, reperfusion damage or metastasis (Frenette & Wagner, 1996; Mojcik & Shevach, 1997; Gonzalez-Amaro et al., 1998).

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Phagocytosis and Inflammation When inflammation remains unresolved, stimulated phagocytes can inflict damage on tissues and produce superoxide anion (0-2) which in turn forms extremely toxic metabolites such as the hydroxyl radical (O-H), peroxy radical (O-OH) hypochlorous acid, hydrogen peroxide, singlet oxygen and hydroperoxide (Rowe et al., 1983). All these oxygen species derived from superoxide are damaging either by a powerful, direct oxidizing action or indirectly as with the hydroxyl and perhydroxyl radical which initiates lipid per oxidation and consequently results in membrane destruction (Mead, 1976). Tissue damage provokes inflammatory responses by the production of mediators and chemotactic factors. Nature has programmed phagocytes such as the neutrophil to use these toxic oxygen metabolites to kill invading organisms. Phagocytic cell attaches itselfto the foreign particle at certain specific sites. These sites also have an adjacent enzyme system known as NADPH-oxidase (Roos et al., 1976) which converts oxygen to superoxide anion which in turn is converted to other toxic oxygen species. Moreover, phagocytes are not particularly sensitive in identifying microorganisms. In general, they will attach to any surface which they are unable to recognize as 'self. In rheumatoid arthritis, the invading neutrophils attach themselves to collagen fibrils in cartilage and other joint tissues (Bromley et al., 1984; Crisp et al., 1984). These fibrils have been made 'non self to the neutrophils by considerable biochemical damage inflicted during the inflammatory process. The phagocyte is physically incapable of engulfing these large structures but attempts to do so. A vesicle is opened up on the surface of the phagocyte but the massive size of the collagen prevents the vesicle from closing and it remains open to the extracellular compartment. However, the cell is stimulated and lysosomes fuse with the vesicle and superoxide anion and other oxygen products are produced. These products bathe the collagen fibrils and other structures with active oxygen species and hydrolytic enzymes. More tissue is rendered 'non-self by the damage inflicted. Consequently more phagocytes are attracted by chemotaxis in an attempt to remove it and a cascade of destruction is initiated. The nonself material is also the source of auto-antigens, characteristic of chronic inflammation (Scher et al., 1980). Therefore, the blood of patients with rheumatoid arthritis contains large number of antibodies which have been formed to act against auto-antigens produced by enzymic or oxygen damage. A characteristic antibody is rheumatoid factor is formed which is an antibody formed against altered immunoglobulins (IgG). Another common antibody formed is antinuclear antibody (ANA), formed against antigens produced from cells disrupted in the disease process. The phenomenon of frustrated phagocytosis is a key pathological event in the transformation of acute inflammation to chronic inflammation.

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CONCLUSIONS The process of inflammation is very complex; different scientists have given different views regarding inflammation and its propagation. To understand the complexity of this process we tried to explain some of the mediator mechanisms of this protective defence of the body. We feel that this review shell give some understanding to the readers about the basis of inflammation and its mediators responsible for its initiation and activation yet this is not the ultimate according to our views but some of the achievements of the science towards inflammation. Although there are still so many things left unwound to know completely about inflammation yet a sincere attempt has been made to explain the phenomenon of inflammation and its mediators.

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Wissman, I.L. and Cooper, M.D. (1993). How the immune system develops. Sci. Am., 269(3): 33-39. Xie, Q.W., Cho, H.J., Calayca, Y.J., Mumford, R.A., Swiderek, KM., Lee, T.D., Ding, A., Troso, T. and Nathah, C. (1992). Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science, 256: 225-228.

7 Recommendations for Reporting Randomized Controlled Trials of Herbal Interventions: CONSORT for Herbal Medicine Trials JOEL J. GAGNIER N.D.l*, HEATHER BOON 2, PAULA ROCHON 3 , DAVID MOHER4, JOANNE BARNEs5 AND CLAIRE BOMBARDIER6 FOR THE CONSORT GROUP

ABSTRACT Controlled trials that utilize randomized allocation are the best tool to control for bias and confounding in trials testing clinical interventions. Investigators must be sure to include in reports of these trials information that is required by the reader to judge the validity and implications of the findings. In part, complete reporting of trials will allow clinician's to modify their clinical practice to reflect current evidence towards the improvement of clinical outcomes. The CONSORT statement was developed to assist investigators, authors, reviewers and editors on the necessary information to be included in reports ofcontrolled clinical trials. The CONSORT statement is applicable to any intervention, including herbal medicinal products. Controlled trials of herbal interventions do not adequately report the information suggested in CONSORT. Recently, reporting recommendations were developed in which several CONSORT items were elaborated to become relevant and complete for randomized controlled trials of herbal medicines. We expect 1. 5955 Ontario St, Unit 307, Windsor, Ontario, Canada, N8S1W6. 2. Leslie Dan Faculty of Pharmacy, 19 Russell Street, University of Toronto, Toronto, Ontario, Canada, M5S 2S2. 3. Baycrest Centre, 3560 Bathurst Street, Toronto, Ontario, Canada, M6A 2El. 4. Children's Hospital of Eastern Ontario Research Institute, 401 Smyth Road, Rm. 210, Ottawa, Ontario, Canada, K1H 8L1. 5. School of Pharmacy, University of Aukland. Position:. Associate Professor in Herbal Medicines. 6. Institute for Work and Health, 481 University Avenue, Suite 800, Toronto, Ontario, Canada, M5G 2E9. * Corresponding author: E-mail: j.gagnie~toronto.ca

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that these recommendations will lead to more complete and accurate reporting of herbal trials. We wrote this explanatory document to outline the rationale for each recommendation and to assist authors in using them by providing the CONSORT items and the associated elaboration, together with examples of good reporting and empirical evidence, where available, for each. These recommendations for the reporting of herbal medicinal products presented here are open to revision as more evidence accumulates and critical comments are collected. Key words : Herbal interventions, randomized controlled trials, CONSORT

INTRODUCTION Randomized controlled trials (RCTs) provide the best evidence for efficacy of health-care interventions (Altman et al., 2001). Carefully planned and well-executed RCTs give us the best estimates of treatment effect and can thusly guide clinical decision making (Mulrow et al., 1998; Sackett et al., 1998). Although, trials which lack methodological rigor cause over- or underestimations oftreatment effect sizes due to bias or confounding factors (Schulz et al., 1995; Moher et al., 1996; Jadad et al., 1996; Begg et al., 1996; Moher et al., 2001; The CONSORT Group). Hence, efforts have been undertaken towards improving the design and reporting of RCTs (Altman et al., 2001; Mulrow et al., 1998; Sackett et al., 1998; Schulz et al., 1995; Moher et al., 1996; J adad et al., 1996; Ioannidis et al., 2004; MacPherson et al., 2001). Current research suggests that reporting quality of complementary and alternative medicine (CAM) trials is poor (Linde et al., 2001a; Moher et al., 2002). Linde et al. (2001a) found that most CAM trials do not describe the generation of the random sequence, an adequate method of allocation concealment, and the number and reasons for drop outs and withdrawals. Moher et al. (2002) (The CONSORT Group, 2004) reported that a sample of pediatric CAM RCTs reported less than 40% of the CONSORT checklist items with a 24% increase in the number of checklist items included in reports over time. That is, less than half of all information necessary in the reporting ofthese trials appeared in their reports. Specifically, only 50% of trials reported how random numbers were generated and 25% if allocation concealment was done (Moher et al., 2002). The results suggest that a large proportion of CAM trials have poor reporting quality resulting in difficulties with assessment of internal and external validity (Linde et al., 200la; Moher et al., 2002). Linde et al. (2001a) showed that reporting quality may vary across different types of complementary therapies with herbal medicine 1 trials

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being somewhat superior to homeopathy and acupuncture trials. Although, several systematic reviews state that trials of botanical medicine still fail to report information necessary to judge internal validity, external validity, and reproducibility (Gagnier et ai., 2003a; Gagnier, 2003b; Little & Parsons, 2000). A study examining the quality of reports of a sample of 206 English language herbal medicine RCTs found that less than 45% of the information suggested within the CONSORT statement was reported (Gagnier et ai., 2006a). For example, approximately 28% of trials described if the person administering the intervention was blinded to group assignment or not, only 22% described the methods for implementing the allocation sequence and 21% the method for generating the allocation sequence. Also, reporting quality differed between individual botanical medicines and improved across decades from the 1980s to the 2000s (Gagnier et ai., 2006a). Furthermore, it has been suggested that trials often do not include detailed information on the herbal product itself (Gagnier et ai., 2003a; Gagnier, 2003b). It is known that herbal medicines may vary by part of plant used, time of harvest, active constituent levels, type of extract (aqueous, alcoholic, glycerin) and delivery form. Therefore, results of clinical trials on heterogeneous products may vary considerably even if they are using the same botanical species. Variation in herbal products between trials precludes pooling in systematic reviews of herbal medicines since invalid inferences may result from the combined data (Gagnier et ai., 2003a; Gagnier, 2003b; Linde et ai., 2001b). It is clear that readers, editors and reviewers require increased transparency in the reporting of RCTs of botanical medicines. Reporting guidelines for controlled clinical trials have been developed.

The consolidated standards of reporting trials (CONSORT) statement was first published in 1996 and revised in 2001 (Begg et ai., 1996; Moher et ai., 2001). This statement consists ofa checklist and flow diagram to guide writers and reviewers on the information that should be available from published reports of two-group parallel randomized controlled trials (RCTs) (Begg et ai., 1996; Moher et ai., 2001). The CONSORT statement has been endorsed by many leading medical journals, editorial associations, professional societies and funding agencies (The CONSORT Group, 2004). Since its inception, several extensions of the CONSORT statement have been developed (Campbell et ai., 2004; Ioannidis et ai., 2004). Recently CONSORT was extended to cluster randomized trials (Campbell et ai., 2004) and for trials of harms Ooannidis et ai., 2004). Also, an international group of acupuncture researchers developed a set of recommendations for

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improving reporting of the interventions in parallel group trials of acupuncture: the Standards for Reporting Interventions in Controlled Trials of Acupuncture or STRICTA (MacPherson et at., 2001). Though not a formal extension of CONSORT, MacPherson et at. (2001) describe STRICTA as a elaboration of item 4 in CONSORT and suggest STRICTA be used together with CONSORT in reporting acupuncture trials. In June 2004 an international group of trialists', methodologists, pharmacologists and pharmacognosists met for a consensus meeting in Toronto, Canada that led to the development of recommendations for the reporting of herbal medicine trials (Gagnier et at., 2006b; See Tables 1 & 2). The resulting guidelines amounted to a set of elaborations of current CONSORT items that will aid editors and reviewers in assessing the internal! external validity and reproducibility of herbal medicine trials, allowing an accurate assessment of safety and efficacy. During the development of the elaborations it became clear that an explanation of the concepts within and underlying the elaborations would aid researchers in planning, conducting and writing reports ofRCTs of herbal medicines. In the current paper we discuss the rationale and scientific background for each elaboration, provide examples of good reporting for each. Where possible, we discuss empirical evidence for each. It should be noted that each elaboration is an addition to existing CONSORT recommendations. Thus all CONSORT items are first listed in Table 1, and elaborations in Tables 2. Table 1. The CONSORT checklist (1,8) Standard Standard CONSORT CONSORT checklist: Paper checklist: Section and Topic Item Title & Abstract

Descriptor

Reported on Page number

How participants were allocated to interventions (e.g. "random allocation," "Randomized" or "randomly assigned"). Scientific background and explanation of the rationale.

Introduction Background

2

Methods Participants

3

Ehgibility criteria for participants and the settings and locations where the data were collected.

Interventions

4

Precise details of the interventions intended for each group and how and when they were actually administered.

Objectives

5

Specific objectives and hypotheses.

6

Clearly defined primary and secondary outcome measures and, when applicable, any methods used to enhance the quality of measurements (e.g. multiple observations, trainmg of assessors).

Outcomes

Table 1. Contd.

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Table 1. Contd. Standard Standard CONSORT CONSORT checklist: Paper checklist: Section and Topic Item Sample size

7

Randomization Sequence allocation Allocation concealment

8

ImplementatIOn

10

Blindmg (Masking)

11

Statistical methods

12

Results Participant flow

13

Recruitment

14

Baseline data

15

Numbers analyzed

16

Outcomes Estimation

and

17

Ancillary analyses

18

9

Descriptor

Reported on Page number

How sample size was determined and, when applicable, explanation of any interim analyses and stopping rules. Method used to generate the random allocation sequence, includmg details of any restriction (e.g., blocking, stratification). Method used to Implement the random allocation sequence (e.g. numbered containers or central telephone), clarifying whether the sequence was concealed until interventIOns were assigned. Who generated the allocation sequence, who enrolled participants, and who assIgned participants to their groups. Whether or not participants, those administering the interventions, and those assessing the outcomes were blinded to group assIgnment. When relevant, how the success ofbhnding was evaluated. Statistical methods used to compare groups for primary outcome(s); Methods for additional analyses, such as subgroup analyses and adjusted analyses. Flow of participants through each stage (a dIagram is strongly recommended). Specifically, for each group report the numbers of participants randomly assIgned, receiving intended treatment, completing the study protocol, and analyzed for the primary outcome. Describe protocol deviations from study as planned, together WIth reasons. Dates defining the periods of recruItment and followup. Baseline demographic and clinical characterIstics of each group. Number of partiCIpants (denominator) in each group included in each analysis and whether the analysis was by "intention-to-treat". State the results in absolute numbers when feasible (e.g. 10120, not 50%). For each primary and secondary outcome, a summary of results for each group, and the estimated effect size and its precIsion (e.g 95% confidence interval). Address mUltiplicity by reportmg any other analyses performed, including subgroup analyses and adjusted analyses, indicating those pre-specified and those exploratory.

Table 1. Contd.

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Table 1. Contd. Descriptor

Standard Standard CONSORT CONSORT checklist: Paper checklist: Section and Topic Item

Reported on Page number

Adverse events

19

All important adverse events or side effects in each mtervention group.

Discussion Interpretation

20

Interpretation of results, taking into account study hypotheses, sources of potential bias or imprecision, and the dangers associated with multiplicity of analyses and outcomes.

Generalisability

21

Generalizabtlity (external validity) of trial results.

Overall evidence

22

General mterpretation of the results in the context of current evidence.

Table 2. Proposed elaborations of CONSORT items for randomized controlled trials of herbal medicine interventions*(20) Standard Standard CONSORT CONSORT checklist: Paper checklist: Section and Topic Item

Descriptor

Reported on Page number

How participants were allocated to interventions (e.g. "random allocation," "Randomized" or "randomly assigned"). Either the title or abstract. or both should state the herbal medicinal product s Latin binomial. the part of the plant lIsed. and the type ofpreparation.

Title & Abstract

Introduction Background

2

Scientific background and explanation of the rationale. Including a brief statement of reasons for the trial with reference to the specific herbal medicinal product being tested and. if applicable. whether new or traditional indications are being investigated

Methods Participants

3

Eligibility criteria for participants and the settings and locations where the data were collected. If a traditional indication is being tested. a description of how the traditional theories and concepts were maintained. For example. participant inclusion criteria should reflect the theories and concepts underlying the traditional indicatIOn.

Table 1. Contd.

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CONSORT for Herbal Medicine Trials

Table 2. Contd. Standard Standard CONSORT CONSORT checklist: Paper checklist: Section and Topic Item InterventIOns

4

4.A. Herbal medicinal product name

Descriptor

Reported on Page number

Precise details of the interventions intended for each group and how and when they were actually administered. 1. The Latm binomial name together with botanical authority and family name for each herbal ingredient; common name(s) should also be included 2. The proprietary product name (i.e. brand name) or the extract name (e.g. EGb-76I) and the name of the manufacturer of the product 3 Whether the product used IS authorized (licensed. registered) in the country in which the study was conducted

1. The part(s) of plant used to produce the product or extract. 2. The type of product used [e.g raw (fresh or dry). extract] 3. The type and concentration of extraction solvent used (e.g., 80% ethanol, H20 IOO%, 90% glycerine etc.) and the herbal drug to extract ratio (drug:extract; e.g. 2'1) 4 The method of authentication of raw material (i e. how done and by whom) and the lot number of the raw material. State if a voucher specimen (I.e. retention sample) was retamed and, if so, where it is kept or deposited. and the reference number. 1. The dosage of the product, the duratIOn of 4.C. Dosage administration and how these were determined. regimen and 2. The content (e.g. as weight, concentration; may be quantitative given as range where appropriate) of all quantified descnption herbal product constituents, both native and added, per dosage unit form. Added materials, such as binders, fillers. and other excipients; e.g. 17% maltodextnn. 3% silicon dioxide per capsule. should also be listed) 3. For standardized products. the quantify of active/ marker constituents per dosage unit form 4.0. 1. Product's chemical fingerprint and methods used Qualitative (equipment and chemical reference standards) and who testing pe/formed it (e.g. the name of the laboratory used). Whether or not a sample of the product (i.e retention sample) was retamed and if so, where it is kept or deposited.

4.B. Characteristics of the herbal product

Table 1. Contd.

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Table 2. Contd. Standard Standard CONSORT CONSORT checklist: Paper checklist: Section and Topic Item

Objectives Outcomes

4.E. Placebo! control group 4.F. Practitioner S 6

Sample size

7

Randomization Sequence allocation

8

Allocation concealment

9

Implementation

10

Blinding (Masking)

II

Statistical methods

12

Descriptor

Reported on Page number

2. Description of any special testing/purity testing (e.g. heavy metal or other contaminant testing) undertaken. Which unwanted components were removed and how (i.e. methods). 3. Standardization' what to (e.g. which chemical component(s) of the product) and how (e g. chemical processes, or biological/fimctional measures of activity) The rationale for the type of control/placebo used.

A description of the practitioners (e.g. training and practice experience) that are a part of the intervention. Specific objectives and hypotheses. Clearly defined primary and secondary outcome measures and, when applicable, any methods used to enhance the quality of measurements (e.g. multiple observations, training of assessors). Outcome measures should reflect the intervention and indications tested considering, where applicable, underlying theories and concepts.

How sample size was determined and, when applicable, explanation of any interim analyses and stopping rules. Method used to generate the random allocation sequence, including details of any restriction (e.g. blocking, stratification). Method used to implement the random allocation sequence (e.g. numbered containers or central telephone), clarifying whether the sequence was concealed until interventions were assigned. Who generated the allocation sequence, who enrolled participants, and who assigned participants to their groups. Whether or not participants, those admmistering the interventions, and those assessing the outcomes were blinded to group assignment. When relevant, how the success of blinding was evaluated. Statistical methods used to compare groups for primary outcome(s); Methods for additional analyses, such as subgroup analyses and adjusted analyses.

Table 1. Contd.

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Table 2. Contd. Standard Standard CONSORT CONSORT checklist: Paper checklist: Section and Topic Item Results Participant flow

13

Recruitment

14

Baseline data

IS

Numbers analyzed

16

Outcomes Estimation

and

17

Ancillary analyses

18

Adverse events

19

Discussion Interpretation

20

Generalisabihty

21

Overall evidence

22

Descriptor

Reported on Page number

Flow of participants through each stage (a diagram is strongly recommended). Specifically, for each group report the numbers of participants randomly assigned, receiving intended treatment, completing the study protocol, and analyzed for the pnmary outcome. Describe protocol deviations from study as planned, together with reasons. Dates defining the periods of recruitment and followup. Baseline demographic and clinical characteristics of each group.lncluding concomitant medication, herbal and complementary medicine use. Number of participants (denommator) in each group included in each analysis and whether the analysis was by "intention-to-treat". State the results in absolute numbers when feasible (e.g. 10/20, not 50%). For each primary and secondary outcome, a summary of results for each group, and the estimated effect size and its precision (e.g. 95% confidence mterval). Address multiplicity by reportmg any other analyses performed, including subgroup analyses and adjusted analyses, indicatmg those pre-specified and those exploratory. All important adverse events or side effects in each intervention group. Interpretation of results, taking into account study hypotheses, sources of potential bias or imprecision, and the dangers associated with multiplicity of analyses and outcomes. Interpretation of the results in light of the product and dosage regimen used. Generalizability (external validity) oftrial results. Where possible, discuss how the herbal product and dosage regimen used relate to what is used in self-care and/or practice. General interpretation of the results in the context of current evidence. Discussion of the trial results in relation to trials of other available products.

*CONSORT items (1,17) are listed In normal text. Proposed recommendations for reports of herbal medic me RCTs are listed in italicized text.

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CONSORT ELABORATIONS INTERVENTIONS

FOR TRIALS

OF

HERBAL

Where necessary, we have elaborated items, to describe information suggested in reports of RCTs of herbal medicinal product interventions. When reporting an RCT of an herbal medicine, authors should consider reporting the information outlined in the CONSORT statement in addition to the information suggested in these elaborations. Below we list original CONSORT items (in normal type) followed by elaborations (in italicized text) for RCTs of herbal interventions. Excerpts from trials include information suggested by the CONSORT items and the developed elaborations.

TITLE AND ABSTRACT Item 1. How participants were allocated to interventions (e.g. "random allocation," "Randomized" or "randomly assigned"). Either the title or abstract, or both should state the herbal medicinal product's Latin binomial, the part of the plant used, and the type of preparation. Examples Title: "A double-blind, placebo-controlled, randomized trial of Ginkgo biloba extract EGb 761 in a sample of cognitively intact older adults: neuropsychological findings" (Mix & Crews, 2002) Abstract: "This was a randomized, double-blind placebo controlled ... The active treatment group received tablets containing 300 mg of Garlic Powder (Kwai) ..... This is equivalent to approximately 2.7 g or approximately 1 clove of fresh Garlic per day." (Isaacshon et al., 1998).

Explanation CONSORT item #1 is meant to aid in the indexing and identification of reports of randomized controlled trials (RCT) using electronic databases (Altman et al., 2001). Hence the use of the words "randomized", "randomly" or "random allocation" is suggested in the CONSORT statement. Additional language is required in the title and abstracts of trials of herbal medicinal products. The practice of evidence-based herbal medicine requires access to the herbal scientific literature. The identification of RCTs of herbal medicinal products requires that the product's Latin binomial, the part of the plant used and the type of preparation be reported in the title and/or abstract. This information would allow increased specificity in the indexing and identification of RCTs of particular herbal medicine products or preparations. Some herbal medicinal products have a specific trade name or commercial name. Where applicable, this name should be listed, together with the Latin binomial of the ingredient herb. When the herbal medicinal product used in the trial is a combination herbal product, we suggest listing

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the product name in the title and the separate herbal medicinal species contained within this product in the abstract. In this way, the title of the trial will not be prohibitively long by listing all separate herbal species' Latin binomials. Studies indicate that searching for complementary and alternative medicine (CAM) related topics is challenging due to diversity of use of controlled vocabulary and indexing procedures between different databases (Murphy et al., 2003). It has been suggested that if authors of CAM trials (e.g. botanical medicine trials) report abstracts or titles without reference to standard controlled vocabulary, indexers may not assign appropriate indexing terms for particular studies (Murphy et al., 2003; McGregor, 2002). A further problem arises from indexers not having a sufficient number and variety of descriptors for CAM interventions (Kronenberg et al., 2001). When reporting RCTs of herbal medicinal products, the use of the information suggested for titles and abstracts above will likely lead to improved indexing and retrieval.

INTRODUCTION Background

Item 2. Scientific background and explanation of the rationale. Including a brief statement of reasons for the trial with reference to the specific herbal medicinal product being used, and if applicable, whether new or traditional indications are being tested. ExampleA The extract of Ginkgo biloba leaves entitled EGb 761 is a complex mixture that is standardized with respect to its flavonol glycoside (24%) and terpene lactone (6%) content [1,2]. These two classes of compounds have been implicated in the benefcial effects of EGb 761 in treating peripheral and cerebral vascular insuffciency, age-associated cerebral impairment, and hypoxic or ischemic syndromes [1,3]. Electron spin resonance (ESR) studies conducted in vitro have shown that EGb 761 is an efficient scavenger of various reactive oxygen species, including superoxide anion radical (0282) and hydroxyl radical (H08), and that it also exhibits superoxide dismutase-like activity [4]. Recent in vitro studies with animal models have revealed that the extract may exert an anti-free radical action in myocardial ischemia-reperfusion injury. In these studies [5,6], inclusion of 200 mg/l of EGb 761 in the medium that was used to perfuse isolated ischemic rat hearts signifcantly improved postischemic recovery, reduced ventricular arrhythmias and enzyme leakage, and lowered the content of A Please

note, all references listed as numbers in all quotes provided refer to the reference number in the original publication from which this excerpt was taken.

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spin-trapped oxyradicals in the coronary effluents. Interestingly, antiarrhythmic effects were also observed when animals were treated orally with EGb 761 prior to heart perfusion, but a significant reduction in ventricular arrhythmias could be achieved only with high dosages (.100 mg/kg for 10 days) [5]. In addition to these studies conducted with EGb 761 [5,6], numerous other studies with experimental animals have indicated that active reduced forms of molecular oxygen, including 0282, HOS, and hydrogen peroxide (H202), are involved in the pathogenesis of tissue injury that follows myocardial ischemia-reperfusion [7-10] (Pietri et al., 1997) ........................ In the present double-blind study, we tested the cardioprotective efficacy of oral treatment with EGb 761, which is known to have in vitro antioxidant properties [4-6], in patients undergoing CPB surgery by manual palpation (Pietri et al., 1997).

Explanation The background of reports of controlled clinical trials partially serve to layout the rationale of the trial (Altman et al., 2001) with special reference to the specific intervention under study. There is great heterogeneity in the types of herbal medicinal products available. Two different preparations/ products of the same herbal species can have different phytochemical profiles, differing pharmacokinetic properties, etc. Given the variability in products, the rationale should clearly overview the scientific data concerning the specific herbal medicinal product under study (e.g. in the above example EGb 761). Where no clinical trials are available for review, extrapolation from pre-clinical work (i.e. animal studies, observational studies, case reports, known mechanisms) is acceptable. Where no data on the product is available, previous research on similar products to that being tested in the current trial should be reviewed. This information should be clearly stated and ideally include a description of a systematic review of previous studies utilizing the herbal product (Altman et al., 2001; Savulescu et al., 1996). Also, if the authors are testing a traditional use, a review of the theory and concepts underlying this indication should be reported. Readers with some relevant knowledge of the area should be able to determine the reasoning for the indication. For example, trials of traditional Chinese medicine (TCM) may choose to test a TCM diagnosis (e.g. Liver Blood Deficiency) and not a western diagnosis (Hepatitis). If this is so, the authors should be explicit in their description of why the particular intervention being tested is indicated. For example,

In traditional Chinese medicine, the "Nei-Kuan" acupoint (EH-6, where EH denotes equilibrium envelope of the heart meridian) has been believed to correlate with the function of the heart (Chuang, 1977). Recently, some investigators (Mah et al., 1992; Hsu et al., 1989) observed that

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acupuncture at Nei-Kuan can improve left ventricular function in patients with coronary heart disease (Ho et aI., 1999). Additionally, other data (i.e. clinical trials, animal studies, observational studies, case reports, known/proposed mechanisms) that would aid in creating a rationale, even for this traditional indication, can be described in the background. The assumption is that a rationale can be clearly and explicitly reported and that it may be derived from scientific, empirical, historica1 4 or traditiona1 5 sources.

METHODS Participants

Item 3. Eligibility criteria for participants and the settings and locations where the data were collected. If a traditional indication is being tested, a description of how the traditional theories and concepts were maintained. For example, participant inclusion criteria should reflect the theories and concepts underlying the traditional indication.

Example There were altogether 118 cases, which were randomly divided into two groups: The QTG (Qingluo Tongbi Granules: A Chinese herbal medicine) treated group and the control group treated with tripteryguim glycosides. In the treated group (n=63), there were 18 males and 45 females, aged from 18 to 65 years with an average of 39.5 ± 16.6 and the disease course ranging 2-22 years averaging 7.5 ± 3.6 years. The cases were graded by xray according to the criteria set by the American Association of Rheumatoid Arthritis (ARA), USA: 7cases were grade I, 30 grade II, and 26 grade III. In the control group (n=55), there were 10 males and 45 females, aged from 18 to 65 years with an average of 38.3 ± 16.7 and the disease course ranging 1-21 years, averaging 6.9 ± 3.1 years. Among them, 15 cases were grade I, 21 grade II, and 19 grade III. The cases in the two groups were comparable in sex, age, disease course and X-ray grading (p>0.05). Diagnostic criteria and TCM differentiation criteria: Diagnosis of RA was made according to the ARA criteria revised in 1987. Criteria for TCM differentiation of the type of yin-deficiency and heat in collaterals: burning pain in joints, local swelling, or deformity and rigidity, reddened skin with a hot sensation, low fever, dry mouth, yellow urine, red or dark red tongue with ecchymosis and petechia, thin or yellow and greasy or scanty fur with fissures, fine, rapid and slippery or fine and rapid pulse. Included in the study were inpatients and outpatients who were diagnosed to have RA of the type of yin-deficiency and heat in the collaterals (Italicized words added from a previous passage in the manuscript) (Xueping et al., 2004).

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Explanation The external validity, generalizability, of a trial is partially dependent on the eligibility criteria for participants (Altman et al., 2001). Reporting of eligibility criteria in trials of herbal medicine interventions is often poor. One study found that less than 75% of RCTs of herbal interventions adequately reported eligibility criteria (Gagnier et al., 2006a). As a result, determination of generalizability of one quarter of these trials would not be possible from reading the published reports. Trials of herbal medicines that aim to test traditional indications must be sure to report any eligibility criteria which reflect this. On a related note, authors may choose to exclude participants with previous use of the specific herbal medicinal product itself. It has been suggested that use of herbal medicinal products prior to trial commencement can lead to increased amounts of adverse effects. A trial of Tanacetum parthenium (Feverfew) (Johnson, 1984) against placebo for migraine prophylaxis included current use of feverfew as an eligibility criterion. Those in the placebo group experienced more side effects, these were attributed to a "post-feverfew syndrome" which is the equivalent of withdrawal effects. Such symptomatic worsening following cessation oflongterm feverfew consumption has been reported elsewhere (Johnson, 1984). To date there is no empirical evidence to suggest that use of an herbal medicine prior to a controlled clinical trial of that same herbal medication biases estimates of treatment effect. Although, the use and reporting of eligibility criteria to exclude trial participants with recent use is suggested.

Example Anyone with a prior adequate trial of St John's wort (at least 450 mg/d) for the treatment of depression or those who had taken St John's wort for any reason in the last month were excluded. To reduce the potential for including a treatment nonresponsive sample, participants who had failed to respond to a trial of an antidepressant (fluoxetine hydrochloride, 20 mg/d, for at least 4 weeks or the equivalent) in the current episode or who had failed to respond to more than 1 adequate trial of antidepressant in a previous episode also were excluded (Shelton et al., 2001).

Explanation It is important that trials of herbal medicines report the settings and locations where the data where collected (Altman et al., 2001). The location highlights physical factors (e.g. climate, food sources), economics, geography and social and cultural factors that may affect the generalizability of a study. As well, research settings may vary greatly in their organization, resources, experience, baselines risk, and physical appearances (Altman et al., 2001). One study found that less than 40% of herbal medicine trial reports adequately reported the setting and location of the trial (Gagnier

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et al., 2006a). External generalizability of trial results partially rests on complete reporting of this information. Item 4: Precise details of the interventions intended for each group and how and when they were actually administered. Where applicable, the description of a herbal intervention should include: 4.A.

Herbal medicinal product name

1. The Latin binomial name together with botanical authority and family name for each herbal ingredient; common name(s) should also be included 2. The proprietary product name (i.e. brand name) or the extract name (e.g. EGb-761) and the name of the manufacturer of the product 3. Whether the product used is authorized (licensed, registered) in the country in which the study was conducted

4.B. Characteristics of the herbal product

1. The part(s) of plant used to produce the product or extract. 2. The type of product used [e.g. raw (fresh or dry), extract] 3. The type and concentration of extraction solvent used (e.g. 80% ethanol, H 2 0 100%, 90% glycerine etc.) and the herbal drug to extract ratio (drug:extract; e.g. 2:1) 4. The method of authentication of raw material (i.e. how done and by whom) and the lot number of the raw material. State if a voucher specimen (i.e. retention sample) was retained and, if so, where it is kept or deposited, and the reference number.

4.C. Dosage regimen and quantitative description

1. The dosage of the product, the duration of administration and how these were determined. 2. The content (e.g. as weight, concentration; may be given as range where appropriate) of all quantified herbal product constituents, both native and added, per dosage unit form. Added materials, such as binders, fillers, and other excipients; e.g. 17% maltodextrin, 3% silicon dioxide per capsule, should also be listed) 3. For standardized products, the quantity of active / marker constituents per dosage unit form

4.D. Qualitative testing

1. Product's chemical fingerprint and methods used (equipment and chemical reference standards) and who performed it (e.g. the name of the laboratory used). Whether or not a sample of the product (i.e. retention sample) was retained and if so, where it is kept or deposited. 2. Description of any special testing/purity testing (e.g. heavy metal or other contaminant testing) undertaken. Which unwanted components were removed and how (i.e. methods). 3. Standardization: what to (e.g. which chemical component(s) of the product) and how (e.g. chemical processes, or biological/ functional measures of activity)

4.E. Placebo/control group

The rationale for the type of control/placebo used.

4.F.

A description of the practitioners (e.g., training and practice experience) that are a part of the intervention.

Practitioner

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The type of information that is required for a complete description of any intervention is relative to the type of intervention being tested. For trials of surgical interventions, for example, a complete description of the individual performing the surgery may be required (Altman et al., 2001; Roberts, 1999). For herbal medicines, the above information is required to determine, with specificity, the key characteristics of the product that was used. A complete description of the product will allow determination of its' efficacy and safety relative to other products. There are a wide variety of commercially available products containing herbal medicines (Dreikorn et al., 1998; Dreikorn et al., 2001; Harkey et al., 2002). In addition there is great variability in the content of these products (Harkey et al., 2002; Gurley et al., 2000; Liberti & Der Marderosian, 1978; Groenewegen & Heptinstall 1986; Heptinstall et al., 1992; Nelson et al., 2002; Zhang et al., 2002). Often products do not contain the amount (weight, volume, proportion) of the individual constituents listed on their label (Manning & Roberts, 2003) or any of the constituents at all. Products containing the same botanical species [e.g. Hypericum perforatum (St. John's wort)] often contain varying amounts of the plant's marker/active 6 constituents. For example, research has shown that commercial products of the following botanical species contain varying levels of their respective constituents: Hypericum perforatum (St. John's wort) (Garrara et al., 2003; Schulte-Lobbert et al., 2004), Camellia sinensis (Green tea) (Manning & Roberts, 2003), Tanacetum parthenium (Feverfew) (Gronewegen and Heptinstall, 1986; Heptinstall et al., 1992; Nelson et al., 2002), Eleutherococcus senticosis (Siberian ginseng), Panax quinquefolius (American/Canadian ginseng) (Harkey et al., 2002), Hydrastis canadensis (Goldenseal) (Weber et al., 2003), and Paullinia cupana (Guarana) (Carlson & Thompson, 1998). As a result, the pharmacological properties and in vitro activities may vary between different products (e.g. Dreikorn et al., 2001; Lefebvre et al., 2004). Also, some studies have shown that botanical products not only contain varying beneficial constituents, but also varying harmful ones (Haller et al., 2004; Oberlies et al., 2004). Therefore, it is necessary that authors of trials of herbal interventions completely describe the product used.

Herbal medicinal product name Example The AG (American Ginseng) capsules contained 3-y-old Ontario grown, dried, ground AG root (Panax quinqueefolius L.) supplied by the same supplier, Chai-Na-Ta Corp., BC, Canada, ..... This AG was the same commercially available product, but from a different batch than the original (Sievenpiper et al., 2003).

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Explanation

Reports should state the Latin binomial and common name/names together with the authority and family name. Reporting of the Latin binomial and common name were also suggested in item 1. The accepted international code of botanical nomenclature (Greuter et al., 1999) indicates that the scientific naming of botanical species must include a Latin binomial (genus and specific epithet) and the authority name. For example, Genus: Taraxicum, Epithet: officinale Authority: Linnaeus. In full, this would result in: Taraxicum officinale L. (Linnaeus is abbreviated as L. here). The authority identifies who originally described the plant. Common names should also be listed here (e.g. Dandelion, Feverfew, St. John's wort). Alone, common names are not sufficient since different herbal species may have the same common name. For example, Echinacea is a common name used for Echinacea angustifolia, Echinacea pallida, and Echinacea purpura. These plants have heterogeneous biochemical profiles (Pellati et al., 2004). If relevant, the proprietary product7 name (i.e. brand name, e.g. Kwai) or the extractS name (e.g. LI 160) and the manufacturer of the product should be reported. Such names are a quick means of identification of the specific herbal product including its contents and manufacturing or production. Alone, these names are not sufficient for the product description. Authors should also report whether the product is licensed in the region where the trial took place. Specific regulatory bodies award licenses for herbal medicinal products. The regulations for attaining licensing are variable across jurisdictions. Though licensing does not ensure product quality or provide the reader with a sufficient description of the herbal product, it does allow the reader to determine the regulatory status and availability of a specific herbal medicine.

Physical characteristics of the herbal product Example (raw herb)

The ginseng capsules contained 3-y-old Ontario dried and ground ginseng root (P. quinquefolius L.) ...... All ginseng and placebo capsules came from the same lot (Vuksan et al., 2001). Example (extract)

... all patients received 1 infusion/day with Ginkgo special extract Egb 761 (batch number: 5242) over 30 - 60 min (1 dry vial in 500 mL isotonic solution). The dry vials contained 200 mg of dry extract from Ginkgo biloba leaves (drug-extract ratio 50 : 1), .... :12 H 20 in 3 mL solution served as solvent (Morgenstern & Biermann, 2003). Explanation

There must be a complete description of the physical characteristics of the herbal product including the partes) of the plant contained in the product or

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extract and the type of product [e.g. raw (fresh or dried) or extract]. The partes) of the plant included in a product are related to the quantities and types of constituents present (Betz et al., 1994). Also, the type of product used should be reported given that different product forms have different types and amounts of constituents (Patora et al., 2003). If the product is an extract, the type and concentration of the extraction solvent should be reported (e.g. 80% Alcohol, H 20 100%, 90% glycerine etc.) as well as the plant to plant extract ratio (plant: plant extract; e.g. 2:1). This ratio tells the reader how much of the starting plant material (either by weight or volume) was required to produce a specific amount ofthe finished extract. The method of authentication of raw material (i.e. how done and by whom) describes how the original material or plant was identified and allows the reader to determine, to some degree, if the raw material for the herbal product was produced from the plant as reported. The lot number of the raw material provides the reader with key information as to where the raw material came from.

Dosage regimen and quantitative description

Example The treatment was provided as 252 tablets containing 50 mg of either Ginkgo biloba standardized extract LI 1370 (containing 25% flavanoids, 3% ginkgolides, and 5% bilobalides) or placebo (both provided by Lichtwer Pharma). Participants were instructed to take three tablets daily for 12 weeks. The extract and dose of Ginkgo biloba were chosen on the basis of the results of previous trials in which this dose of this extract had been reported to be effective in treating cerebral insufficiency> (Drew & Davies, 2001).

Explanation Authors of trials of herbal medicines should report the dosage regimen and provide a quantitative description of the herbal product. Information regarding the dosage and duration of the trial are of great importance to replicating trials, establishing efficacy or harm for specific dosages and durations, and for external generalizibility (Altman et al., 2001). The rationale for dosage and duration of the trial should be clear as unclear reasoning questions the methods of a trial and possibly raises some ethical issues as to why the trial was carried out at all. The weight or amount of all herbal product constituents, both native and added per dosage unit form (i.e. added materials such as binders, fillers, excipients, etc; e.g. 17% maltodextrin, 3% silicium dioxide per capsule) and the percentage of active/marker constituents per dosage unit form (e.g. 0.3% Hypericin per capsule) should also be reported. This provides the reader with a profile of the quantity of the botanical product constituents.

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Qualitative testing Example The content of various ginsenosides (RgI' Re, Rf, Rb, Rc, Rb 2 , and Rd), which are dammarane saponin molecules found among Panax species, was determined in the laboratory of Dr. John T. Arnason at the Department of Biology, Faculty of Science, University of Ottawa, Ontario, Canada, using high-performance liquid chromatography (HPLC) analyses, a method similar to the one developed for the American Botanical Council Ginseng Evaluation Program [27]. A Bechham HPLC system with a reverse-phase Beckham ultrasphere C-18, 5um octadecylsilane, 250 X 4.6mm column was employed for the analyses. The ginsenoside standards used for comparison were provided by two sources. Rg 1 and Re were provided by Dr. H. Fong, University of Illinois and Rf, RbI' Rc, Rb 2 , Rd were provided by Indofine Chemical Co., Somerville N.J. (Vuksan et al., 2000) Table 1. Energy. nutrient, and ginsenodide profile of the American ginseng (Panax quinquefolius L.) and placebo capsules Constituent (per g) E nergy 2 (kJ) 14.68 (kcal) Macronutrients 2 Carbohydrate (g) Fat (g) Protein (g) Ginsenosides (20S)- Protopanaxadiols (%) RbI Rb2

Rc

Rd (20S)-Protopanaxatriols (%) Rgi Re Rf Total (%)

Placebot

Ginsengt

14.39 3.51

3.44

0.73 0.039 0.069

0.57 0.013 0.26

1.53 0.06 0.24 0.44 0.100 0.83

o

3.21

ITo equate energy and macronutrient values to 1, 2, or 3 g American ginseng, multiply by 1, 2, or 3, respectively. To determine values for placebo, multiply by 2. 2Determined by the Association of Official Analytical Chemists methods for macrountrients (18). 3Determined by HPLC analyses (20). Adopted from: Vuksan et al., 200l.

Explanation Trials should report the product's chemical fingerprint 9 and methods used (machinery and chemical reference standards) and who performed it (the

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name ofthe laboratory used). The fingerprint can be reported in a graph or a table describing the key constituents of the herbal medical product. Chemical profiling, using the proper techniques is essential to providing a clear and accurate report of a product's constituents, and provides both qualitative and quantitative information (Sawnson, 2002; Phytomedicine: International Journal of Phytotherapy and Phytopharmacology; German Federal Institute for Drugs and Medical Device, 1999). Bauer and Tittel (1996) have provided some guidelines for the characterization and standardization of plant material used for pharmacological, clinical and toxicological studies. Also the Association of Analytical Communities (AOAC) has outlined standards for analyzing specific herbal medicinal products (AOAC, 2004) as has the American Herbal Pharmacopoeia produced by the American Botanical Council (American Herbal Pharmacopoeia, 2004) and the United States Pharmacopoeia (United states Pharmacopoeia, 2004). Reports might also describe if a voucher specimen 10 (i.e. retention sample) was retained and if so, where it is kept or deposited, so that independent sources can verify the chemical profile. Herbal medicines are often contaminated (Chan, 2003). Thus, a complete description of any special testing/purity testing (e.g. heavy metal or other contaminant testing) and the removal of unwanted components (which and how (the methods» should be included in reports where relevant. All such methods are relevant given that they may alter the composition of the herbal product. Standardization has been hotly debated in the literature (e.g. Swanson, 2002). Often, companies or researchers attempt to standardize botanical products to specific chemical "marker" constituents. These "marker" constituents may be considered to be the primary "active" constituents or merely serve an index of the product's chemical profile (Swanson, 2002). Though products on the commercial market may not be standardized and often an "active" constituent is not known, if standardization was done in a clinical trial of an herbal product, it should be reported. Authors should report, what the product was standardized to (e.g. which chemical component(s», how this was done (i.e. chemical processes, or biological! functional measures l l of activity) (e.g. Murphy et al., 1988) and the percentage of this particular constituent per dosage unit form. The rationale for the type of control/placebo used.

Example The placebo, on the other hand, consisted of identical capsules containing corn flour. The energy, carbohydrate content and appearance ofthe placebo were designed to match that of the AG (American Ginseng capsules). (Italicized words added). (Sievenpiper et al., 2003)

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Explanation In botanical medicine trials, as in other trials, it is important to have a complete description ofthe characteristics of the control group and the way in which it was disguised (Altman et al., 2001). If a placebo control was used, the placebo should be closely matched to the control intervention (Vickers & de Craen, 2000). For trials of herbal interventions the rationale for the type of control/placebo used should be described. There have been some trials that have reported using placebos that are matched to color and smell, but that contain ingredients that are active (e.g. Palevitch et al., 1997). If a control group is active, comparisons between it and the experimental group will be affected. While it may be a challenge to construct matched placebos for certain herbal product interventions, it is not impossible. A description of the practitioners that are a part of the intervention.

Example In order to participate in the study, physicians had to (i) be a medical specialist with a degree in internal medicine and general medicine, (ii) have a certified degree in TCM by a German society for medical acupuncture, and (iii) have at least 5 years of practical experience in TCM (according to the German Acupuncture Societies Working Group standard) ....... The herbal formulations for the TCM group were designed by a herbalist (CarlHermann Hempen) and prepared by a pharmacist who both specialise in Chinese herbal medicine (S. Dietz, Franz-Joseph-Pharmacy, Munich, Germany). In addition to the basic formula, every patient received a second additional formula tailored to his or her individual TCM diagnosis (Table 1). (Brinkhaus et al., 2004)

Explanation On occasion an herbal intervention trial may include a health-care practitioner as part of the intervention. Practitioners have varying levels of training, years of experience, theoretical orientations and work in different environments. Similar to surgical trials, such trials should provide a description of the practitioners (e.g. training and practice experience) that are a part of the intervention (Altman et al., 2001).

Outcomes Item 6: Clearly defined primary and secondary outcome measures and, when applicable, any methods used to enhance the quality of measurements (e.g., multiple observations, training of assessors). Outcome measures should reflect the intervention and indications tested considering, where applicable, underlying theories and concepts.

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Example All outcome measures were assessed at baseline and after 30 days of treatment at the follow-up visit. The primary outcome measures were changes in quality oflife as measured by the Physical and Mental Component Summary scales of the 12-Item Short Form Health Survey (SF-12). The SF-12 is widely used in measuring health and quality of life and has been shown to have a high level of agreement with scores from the original 36Item Short Form Health Survey (SF-36)[11]. The SF-36 has been validated in several Chinese studies, whereas evaluation of the SF-12 is ongoing [11]. Secondary outcome measures included assessments of physical performance, memory, sexual function, and qi ................. The qi scale is a 17-item instrument (14 items on an interviewer-administered questionnaire and three physical examination items) that was developed through an international collaboration of clinical investigators with expertise in scale development and traditional Chinese medicine. Questionnaire items address symptoms commonly included in a traditional Chinese medical interview, including breathing, energy level, appetite, heartburn, sweating, bowel patterns, pain, temperature sensations, sleep habits, and sexual ability. The physical examination items address tongue coating, tongue muscle quality, and pulse quality. The scale was developed for this study and has not been validated. The 14 questionnaire items are scored on a scale of 0 to 4 points, and the physical examination items are scored on a scale of 0 to 3. The total qi score is the sum of each score, ranging from 0 (best) to 65 (worst) (Bent et at., 2003). Explanation As with any RCT, outcome measures, both primary and secondary, should be relevant to the indications being tested be fully reported and describe any methods used to enhance the quality of measurements (1,8)(Altman et al., 2001; Moher et al., 2001). When performing RCTs testing herbal interventions, concepts that go beyond western medical terminology and understanding may be relevant. For example, in the above trial the particular Chinese herbal remedy being tested is purported to increase longevity, quality oflife, energy, memory, sexual function and Qi, a Chinese concept that is loosely translated as vital energy. Therefore, in addition to measures of health and quality of life, these investigators required a measure of Qi to test the change in vital energy during the course of this trial. Ultimately, to test the function of traditional herbal medicines we advise that the outcome measures reflect the underlying theories and concepts and therefore the indications for the specific herbal medicine intervention under investigation.

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RESULTS Baseline data

Item 15: Baseline demographic and clinical characteristics of each group. Including, concomitant medication use or herbal medicinal product use. Example Eight patients (mean age 44.9 (SEM 4.2) years) received feverfew and nine (mean age 51.2 (2.3) years) received placebo capsules. The patients in the active group had taken 2.44 (0.2) small leaves of feverfew daily for 3.38 (0.58) years before entry to the study, and those in the placebo group had taken 2.33 (0.48) small leaves daily for 4.18 (0.67) years. Thus the two groups did not differ in the amount of feverfew consumed daily or the duration of consumption ..... . One patient in each group was taking conjugated equine estrogens (Premarin); the patient in the placebo group was also taking pizotifen. One patient given feverfew was taking the combined oral contraceptive Orlest 21. On patient in each group was taking a diuretic: the patient given feverfew was taking clorazepate and the patient given placebo was also taking a product containing tranylcypromine and trifluorperazine. In addition, two people in the placebo group were taking vitamin preparations and one prochlorperazine (Johnson et al., 1985).

Explanation A complete description of participants who entered a trial allows readers and clinician's to assess how relevant the trial is to a specific patient. As a part of the baseline assessments in trials of herbal medicinal products, authors should clearly assess and describe any current medication or herbal product use. Differences between groups on medication or herbal product use may confound results (Piscitelli & Burstein, 2002).

DISCUSSION Interpretation

Item 20: Interpretation of results, taking into account study hypotheses, sources of potential bias or imprecision, and the dangers associated with multiplicity of analyses and outcomes. Interpretation of the results in light of the product and dosage regimen used. Example Although EGb 761 is generally used at a dose of 120 mg/day in treating chronic disease states, we chose to administer the extract at more than twice its usual dose, but for only 5 days before the operation, to cope with the enhanced generation of oxidant species that was expected to follow

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post-unclamping procedures. Measurements of DMSO/AFR concentrations indicated that EGb 761 treatment significantly protected plasma ascorbate levels in all sampling sites during the initial 5-10 min. ofreperfusion (see Table 3), a period during which free radical processes are considered to be critical. Analyses of plasma TBArs concentrations revealed that EGb 761 treatment also suppressed (or substantially attenuated) the transcardiac release of MDA, indicating protection against free radical-induced lipid peroxidation. These two findings offer some clues regarding the mechanisms that underlie the protective action of EGb 761 in open-heart surgery. It has been reported that EGb 761 protects ischemic rat hearts against reperfusion-induced decreases in ascorbate [6]. We did not observe any in vitro chemical interaction between EGb 761 and AFR or DMSO/AFR that would have maintained ascorbate levels. Therefore, in performing its antioxidant role, it seems that the extract competes effectively with circulating ascorbate (e.g. by directly quenching oxyradicals once they have been formed) ................ (Pietri et al., 1997).

Explanation The discussion section of a clinical trial report provides the reader with information to determine the pros and cons of the study and how this study relates to other research in the area. Understanding how the results with this product and dosage regimen relate to the results of other trials with similar or different products or dosage regimens allows the reader to establish a context from which to determine strengths or drawbacks of the specific intervention used. Therefore, authors should clearly state specific aspects of the product or dosage regimen that could have resulted in trial findings. This concept is discussed here under Item 20, to highlight the necessity of authors of reports of RCTs of herbal products to explicitly consider the product and dosage regimen as potential strengths or drawbacks of the study. Though we have formally separated this from the elaboration on Item 22, discussion of the trial results in the context of the current evidence (Item 22), we admit that these aspects of the discussion section are closely related, and may be written together in the body of the manuscript.

Generalizability Item 21: Generalizability (external validity) of trial results. Where possible, discuss how the herbal product used relates to what is used in self-care and I or practice. Example G 115 is a standardized ginseng extract which is often complexed with various other substances and marketed commercially. Ginsana®, Gericomplex®, Geriatric Pharaton®, and ARM229 are several commercial standardized

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ginseng products which have been studied, and may include some or all of the following substances in addition to G 115: vitamins, minerals, trace elements, dimethylaminoethanol bitarate. A word of caution for the consumer. As noted previously, the FDA classifies ginseng as a food supplement, so it is marketed rather extensively in health food stores. An estimated 5-6 million Americans use ginseng products (6). However Chong and Oberholzer (11) note that there are problems with quality control, and indeed a recent report (12) indicated that of 50 commercial Ginseng preparations assayed, 44 contained concentrations of ginsenosides ranging from 1.9% to 9.0%, while six of the products had no detectable ginsenosides (Dowling et al., 1996).

Explanation Generalizability, or external validity is the extent to which the results of a study hold true in other circumstances (Fletcher et al., 1996). The word "circumstances" here can mean other individuals or groups of individuals, other similar interventions, dosages, timing, administration routes, and other settings for starters. Given the wide variability in herbal medicinal products available on the market, and their variable quality and content, a review of how the products used in the current trial relate to what is available and/or used by consumers and practitioners is quite valuable. This information would allow the reader to determine the availability of products that may act similarly to the one used in the triaL Application of clinical trial results partly relies on the availability of the intervention or a similar intervention.

Item 22: General interpretation of the results in the context of current evidence. General discussion of the trial results in relation to trials of other available products. Example The majority of published studies to date have used a powdered garlic preparation, similar to the preparation method used in this study. Considerable variability in outcomes exists between these studies. For example, Adler et al. [13], using a commercial dehydrated garlic tablet, reported a significant net drop of 13.1% in LDL-C levels relative to the placebo group in 12 weeks, and Jain et al. [15], using the same product and a similar design, reported a significant net decrease of 8% in LDL-C levels in moderately hypercholesterolemic adults. However, three other studies [19,20,22], using the same dosage of the same commercial dehydrated garlic powder product (Kwai®, Lichtwer Pharmaceuticals) reported no significant effect. The dose of powdered garlic tablets used in the five studies just cited, 900 mg:day, was similar to the full dose of 1000 mg:day used in this study. The allicin content of the tablets used in this study, 1500 mg:day in the full dose, was lower than the amount used in other studies with powdered

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garlic preparations. Other types of garlic preparations used in lipid lowering trials have included aged garlic extract and teamed garlic oil. Steiner et al. [14] used a large dose, 9 tablets: day, of aged-garlic extract, and reported a statistically significant 4.6% lowering of plasma LDL-C levels. In contrast, a recent study using steamed garlic oil supplementation reported no significant effect on cholesterol levels in hypercholesterolemic adults after 12 weeks [18]. One explanation could be that the oil is not as effective as dehydrated garlic powder because it contains different sulfur-containing phytochemicals [30]. Some of the discrepancies reported in these studies can be explained by the heterogeneity that exists among them in terms of study design, duration, subject characteristics, adherence, or confounders such as weight, diet, and exercise (Gardner et al., 2001).

Explanation Discussing trial results in the context of relevant studies is important to put the trial results in the context of existing empirical evidence (Altman et al., 2001; Annals ofInternal Medicine, 2001). Some trials fail to provide the reader with sufficient information to determine how the current results relate to other research. For example, Drew and Davies (2001) report that "Ginkgo biloba extract LI 1370 had no greater therapeutic effect than placebo in treating tinnitus. In addition, other symptoms of cerebral insufficiency were not significantly affected by the treatment (Table 3). The results from this trial are similar to some reports and contrast with others. 2 This study differs from other trials in many ways." (p.5). This provides the reader with little information from which to judge the efficacy of the product currently tested relative to other products that have been tested. Different botanical products can have different constituents and therefore differing therapeutic effects (Harkey et al., 2002; Gurley et al., 2000; Liberti & Der Marderosian, 1978; Groenewegen & Heptinstall 1986; Heptinstall et al., 1992; Nelson et al., 2002; Zhang et al., 2002). It is suggested here that discussion sections of trials of botanical interventions include a discussion of the trial results in the context of previous research while offering explicit consideration of the similarities or differences between products used therein. It is inappropriate to support or refute a trial's results by referring to literature that has tested a different product. Authors should be careful to clearly report when they are drawing inferences between heterogeneous products. When clinical trials on the specific product tested do not exist, pre-clinical data should be discussed. This includes animal, in vitro and other data.

COMMENTS Randomized allocation is the best tool to control for bias and confounding in controlled trials testing clinical interventions. Investigators must be sure to include in reports of these trials information that is required by the

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reader to judge the validity and implications of the findings. In part, complete reporting of trials will allow clinician's to accurately appraise studies so as to modifY their clinical practice to reflect current evidence. The CONSORT statement was developed to assist investigators, authors, reviewers and editors on the necessary information to be included in reports of controlled clinical trials. The CONSORT statement is applicable to any intervention, including herbal medicinal products. Controlled trials of herbal medicines interventions do not adequately report the information suggested in CONSORT. Recently, several CONSORT items were elaborated to become relevant and complete for controlled trials of herbal medicines (Gagnier et al., 2006b). We expect that these recommendations will lead to more complete and accurate reporting of herbal medicine trials. We wrote this explanatory document to further explain the suggested elaborations and to assist authors in using them. We provide the CONSORT items and the associated elaborations, together with examples of good reporting and empirical evidence, where available. These recommendations for the reporting of RCTs of herbal medicine are open to change and revision as more evidence accumulates and critical comments are collected.

ACKNOWLEDGEMENTS The contributing papers included several additional authors that were instrumental in the development of concepts included in this chapter: Heather Boon, David Moher, Joanne Barnes, Paula Rochon and Claire Bombardier. We would like to thank Greer PaHoo for aiding in the preparation for the June meeting and Jaime DeMelo and Cyndi Gilbert for assisting Joel Gagnier and Claire Bombardier during the meeting procedures.

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8 Anxiolytic Activity Screening Studies on Extracts of a Few Medicinal Plants

ABSTRACT Aethusa cynapium, Artemisia abrotanum, Cimicifuga racemosa and Clematis erecta have been used traditionally in anxiety and sleep disorders but these plants have not been investigated so far to substantiate these therapeutic claims. The present study was aimed at screening the anxiolytic potential of the four plants. Petroleum ether, chloroform, methanol and water extracts of the plants have been evaluated for their anxiolytic activity using the elevated plus-maze model in mice. The methanol extract of Aethusa cynapium has demonstrated significant anxiolytic activity as compared to the standard drug diazepam. These results indicate that it is worth undertaking in depth investigation on Aethusa cynapium with a view to develop a potentially useful anxiolytic drug. Key words : Aethusa cynapium, Artemisia abrotanum, Cimicifuga racemosa, Clematis erecta, anxiolytic activity

INTRODUCTION Anxiety is a normal emotional response to real or potential danger. If anxiety becomes excessive, out of proportion and interferes with everyday life, management becomes necessary. The conventional treatment of anxiety involves use of medication like benzodiazepines, selective serotonin reuptake inhibitors (SSRls) etc or psychological therapy or a combination 1. Department of Pharmaceutical Sciences and Drug Research, Punjabi University,

Patiala, India. 2. #Pharmacognosy Division, University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh 160014, India (# - Address for correspondence). * Corresponding author : E-mail: [email protected]

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of the two. Common limitations of antianxiety drug therapy include comorbid psychiatric disorders and increase in dose leading to intolerable side effects (Cates et al., 1996). This has led researchers worldwide to investigate plants which are commonly employed in traditional and alternative systems of medicine for sleep disorders and related diseases (Farnsworth, 1980). Various plants have been investigated for their anxiolytic effects (Carlini, 2003). Valeriana species such as V. walliichii, V. officinalis, V. eduli, V. thalictroides have demonstrated antianxiety effect in human beings (Handa, 1992), Piper methysticum is employed clinically for the treatment of anxiety (Pepping, 1999; Reeder & Cupp, 2000; Billia et al., 2002). Numerous plants like Nardostachys jatamansi (Handa, 1992), Passiflora species like P. incarnata (Dhawan et al., 2001, 2001a), P.alata (Petry et al., 2001), Citrus aurantium (Carvalho-Frietas et al., 2002), Scutellaria lateriflora (Awad et al., 2003), Stachys lavandulifolia (Rabbani et al., 2003), Ginkgo biloba (Kuribara et al., 2003), Coriandrum sativum (Emamghoreishi et al., 2005), Turnera aphrodisiaca (Kumar & Sharma, 2005, 2005a), Centella asiatica (Wijeweera et al., 2006), Adiantum tetraphyllum (Bourbonnais-Spear et al., 2007), Apocynum venetum (Grundmann et al., 2007), have shown antianxiety activity in experimental animals. In the present study four plants, used in the traditional and complementary medicine have been investigated for their anxiolytic potential.

Aethusa cynapium Linn. (Umbelliferae) commonly called Fool's Parsley, has been used traditionally for gastrointestinal complaints in children, infantile cholera, summer diarrhoea, convulsions, sleep disorders and delirium (Flemming, 2000). In the homeopathic system of medicine, A. cynapium is used for milk intolerance in children, pylorus cramps and acute diarrhoea with vomiting (Vikramaditya & Joshi, 1997). A. cynapium contains alkaloid (Vikramaditya & Joshi, 1997), polyacetylenes (Andreev et al., 2001), essential oil, flavone glycosides and ascorbic acid (Flemming, 2000). Artemisia abrotanum Linn. (Compositae) commonly called Southernwood (Good, 1974), has been used traditionally for the treatment of digestive ailments, worms, menstrual disorders, marasmus, diarrhoea (www.herbs2000.com, 2000) and to relieve mental tension (www.herbs for life, 2003). A. abrotanum contains antimalarial sesquiterpene isofraxidin coumarin ethers (Greger et al., 1983; Cubukcu et al., 1990), essential oil (Mucciarelli et al., 1995), hydroxy coumarins (Elzbieta et al., 1984), spasmolytic flavonoid glycosides (Bergendorff & Sterner, 1995), peroxysemiketals (Ruecker et al., 1993) and choleretic phenolic acids (Swiatek et al., 1998).

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Cimicifuga racemosa Linn. (Ranunculaceae; synonym Actea racemosa) commonly known as Black Cohosh (Gleason & Cronquist, 1991), has been traditionally used to treat diarrhoea, sore throat, rheumatism, painful menstrual periods, menopausal disorders (Lieberman, 1998) dysmenorrhea, dyspepsia and snakebites (Bisset, 1989). The herb is available commercially in combination with St. John's Wort for depressive moods associated with premenstrual and menopausal symptoms (Fleming, 2000). The tincture of C. racemosa is used as anxiolytic, sedative emmenagogue and antitussive (Vikramaditya & Joshi, 1997). Remifemin® is a standardized extract ofthe plant available in Germany for management of menopause syndrome (Bisset, 1989). The plant is a hormone-free phytomedicine for treatment of climacteric symptoms related to menopause (Freudenstein et al., 2000; Liu et al., 2001; Kennelly, 2002). C. racemosa is rich in triterpene glycosides (Bedir & Khan, 2000; Wende et al., 2001; Chen et al., 2002, 2002a), 15,16-seco-cycloartane glycosides and tetra nor cycloartane glycosides (Nishida et al., 2003, 2003a), phenylpropanoid esters, aromatic acids, isoflavone and metals (Akihisa, 1999; Panossian et al., 2004). Clematis erecta Linn. (Ranunculaceae; synonym Clematis recta) is employed traditionally for disorders of skin, lymphatic system and genitourinary organs especially for gonorrhea. The tincture of C. erecta, an official preparation in the Pharmacopoeia Homeopathica Polyglottia 1872, is employed for the symptomatic relief of anxiety, nervous tension, insomnia, stress, fear, panic, colds, flu, fever and minor injury (Llyod & Lloyd, 1887; Felter & Llyod, 1898). C.erecta is reported to contain quaternary aporphine alkaloid (Slavik et al., 1987, 1995). MATERIALS AND METHODS

Plants Ariel parts ofAethusa cynapium, Artemisia abrotanum, Cimicifuga racemosa and Clematis erecta were collected from a cultivated source (Rati Ram Nursery) at village Khurrammpur via Kalsia, district Saharanpur (U.P., India) in March 2005. The identities of the plants were confirmed at the Museum-cum-Herbarium of the University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh.

Tested material Petroleum ether, chloroform, methanol and water successive Soxhlet extracts of the dried aeriel parts of the four plants viz. Aethusa cynapium, Artemisia abrotanum, Cimicifuga racemosa and Clematis erecta were prepared. Yields (as %, dry weight) are detailed in Table 1.

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Table 1. Percentage yields of extracts of the four investigated plants Plant extract Pet. Ether Chloroform Methanol Water

Aethusa cynapium 2.2 1.8 5.8 2.9

Yields (%, dried weight) Artemisia Clematis abrotanum erecta 1.3 1.3 9.1 3.9

0.5 3.6 12.1 10.3

Cimicifuga racemosa 2.5 2.8 12.8 15.6

Studied Activity Anxiolytic activity was evaluated using modified elevated plus-maze (Montgomery, 1958; Pellow et al., 1985; Kulkarni & Reddy, 1996). Vehicle [5% Tween 80 in Simple Syrup LP. (66.7% w/w sucrose in water)], test extracts and diazepam (reference drug), both suspended in vehicle, were administered orally 45 min prior to the antianxiety test, and the behavior of animals on the elevated plus-maze was recorded for 5 min in terms of (i) number of entries into open or closed arms (ii) average time spent by the mouse in open or closed arms.

Animals Swiss albino mice of either sex, weighing 20-24 g were procured from the Central Animal House of the Panjab University, Chandigarh. The mice were allowed standard laboratory feed and water ad libitum. Groups of five mice were used in all sets of experiments. Animals were fasted overnight before use. All animal studies were carried out as per the guidelines of the Institute Ethical Committee, Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala.

Statistical Analysis Analysis of variance (AN OVA) and the Tukey's multiple range tests was employed to determine statistical significance. Difference were considered significant at p<0.05. RESULTS AND CONCLUSIONS Aethusa cynapium, Artemisia abrotanum, Cimicifuga racemosa and Clematis erecta have been used traditionally in anxiety and sleep disorders but these plants have not been investigated so far to substantiate these therapeutic claims. The present study was carried out to validate the traditional claims. Different extracts of the four plants have been prepared using solvents in increasing order of polarity. This is done in order to extract completely non polar to completely polar constituents from the plant material. The anti anxiety activity of these extracts were compared with

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the activity of the standard drug diazepam and the control using the elevated plus-maze model. The elevated plus-maze, is a novel test for the selective identification of anxiolytic drug effects in rodents (Vogel, 2002). The elevated plus-maze model is effective, cheap and simple, requires no preliminary training for the mice, and does not cause much discomfort to them while handling. The fear due to height (acrophobia) induces anxiety in mice when placed on the elevated plus-maze. The ultimate manifestation of anxiety and fear then is exhibited by decrease in motor activity, which is measured by the time spent by mice in the open arms. Anxiolytic compounds, by decreasing anxiety, increase the open arm exploration time, anxiogenic compounds have the opposite effect (Kulkarni, 2002). The mean time spent by the mice in open arms of the EPM after oral administration of various doses of the different extracts of the four plants is reported in Table 2. Table 2. Anxiolytic effects of extracts of the four investigated plants using the Elevated Plus Maze model Comparison with Average Time spent in open arms (seconds) a Control (Vehicle, 0.25 mL) 3.2 ± 1.3 Standard (Diazepam, 2 mg/kg) 12.2 ± 1.2 Treatment

Average Time spent in open arms (seconds)a Dose (mg!kg), Aethusa Artemisia Cimicifuga Clematis p.o cynapium abrotanum erecta racemosa

Pet.Ether Extract

50 100 200 400

5.7 6.8 7.9 5.9

± ± ± ±

1.0*'** 0.4*·** 0.8*·** 0.9*·**

4.5 3.2 7.9 4.9

± ± ± ±

0.5 *.** 0.4* 0.9*·** 0.5*·**

1.7 1.1 1.6 3.2

± ± ± ±

0.1*·** 0.1*·** 0.2*·** 0.4*

2.3 2.7 3.3 2.6

± ± ± ±

0.2* 0.2* 0.4* 0.2*

Chloroform extract

50 100 200 400

5.1 8.1 7.6 7.0

± ± ± ±

1.2*·** 1.2*'** 1.0*·** 1.3*·**

2.9 4.3 2.6 2

± ± ± ±

0.2* 0.5* 0.2* 0.2*·**

1.0 1.6 1.4 2.1

± ± ± ±

0.1*·** 0.2*·** 0.2*·** 0.2*·**

1.7 2.6 0.8 3.3

± ± ± ±

0.1*·** 0.2* 0.2*·** 0.4*

Methanol extract

50 100 200 400

7.7 8.2 9.5 12.7

± ± ± ±

0.9*·** 0.9*'** 1.1*·** 1.5**

0.9 1.6 1.1 6.9

± ± ± ±

0.1*·** 0.1*·** 0.1*·** 0.7*·**

0.6 1.0 1.9 5.6

± ± ± ±

0.1* 0.1* 0.1* 0.6*

2.6 2.5 4.7 1.2

50 100 200 400

5.2 8.2 8.4 7.9

± ± ± ±

0.6*'** 1.7*·** 1.5*·** 1.4*·**

0.8 0.7 0 8.3

± ± ± ±

0.1*·** 0.1*·** 0.0*·** 1.0*·**

2.2 ± 1.5 ± 2.7 ± 1.15±

0.3* 1.8*·** 0.3* 0.2*·**

0.5 0.9 1.2 0.7

Water extract

± 0.2* ± 0.2*

± 0.5*'** ± 0.1*·** ± ± ± ±

0.1*'** 0.1*·** 0.1*·** 0.1*·**

a:Values are expressed as mean ± S.D. (n = 5). ANOVA followed by Tukey's multiple range test. p<0.05. * =significant with respect to standard, ** = significant with respect to control.

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Animals treated with diazepam (2 mg/kg, p.o.) spent 12.2 sec in open arms of the plus maze and the control group animals spent 3.2 sec. Of the four extracts of Aethusa cynapium, the methanol extract shows a dose dependent increase in motor activity, with the animals spending 12.7 seconds at 400 mg/kg in the open arms of the maze. This activity is statistically comparable to that of diazepam. The Artemisia abrotanum extracts, although significant at some doses when compared to the control, were not significant in comparison with diazepam. The four extracts of Clematis erecta and Cimicifuga racemosa do not show anxiolytic activity at any of the doses administered. The results of the present study show that of the four plants tested only Aethusa cynapium demonstrated significant anxiolytic activity, and indicate that it is worth undertaking in depth investigation on Aethusa cynapium with a view to develop a potentially useful anxiolytic drug.

REFERENCES Akihisa, T., Hideshima, R., Koike, K., Kimura, Y. and Nikaido, T. (1999). Cycloarta-16,24dien-3-ol: revised structure ofcimifugenol, a cycloartane triterpenoid. Chem. Pharm. Bull., 47: 1157- 1160. Andreev, G.N., Schulz, H., Schrader, B., Fuchs, R., Popov, S. and Handjieva, N. (2001). Nondestructive NIR-Ft-Raman analyses in practice. Partl. Analyses of plants and historic textiles. Fresenius J. Anal. Chem., 371(7): 1009-17. Awad, R., Amason, J.T., Trudeau, V., Bergeron, C., Budzinski, J.W., Foster, B.C. and Merali, Z. (2003). Phytochemical and biological analysis of skullcap (Scutellaria latenflora L.): a medicinal plant with anxiolytic properties. Phytomedicine, 10(8): 640-9. Bedir, E. and Khan, l.A.(2000) Cimiracemoside A: A new cyclolanostanol xyloside from the rhizome of Cimic~fuga racemosa. Chem. Pharm. Bull., 48: 425-427. Bergendorff, O. and Sterner, O. (1995). Spasmolytic flavonols from Artemisia abrotanum. Planta Medica, 61(4): 370-71. Billia, A.R., Gallori, S. and Vincieri, F.F. (2002). Kava-kava and anxiety:growing knowledge about the efficacy and safety. Life Sci., 70: 2581-97. Bisset, N.G. (1989). Herbal drugs and phytopharmaceuticals- A handbook for practice on a scientific basis, CRC press, 90-91. Bourbonnais-Spear N., Awad R., Merali, Z., Maquinc, P., Cal, V. and Amason, J.T. (2007). Ethnopharmacological investigation of plants used to treat susto, a folk illness. J. Ethnopharmacol., 109: 380-387. Carlini, E.A. (2003). Plants and the central nervous system. Pharmacol., Biochem. and Behavior, 75: 501-512. Carvalho-Freitas, M.l. and Costa, M. (2002). Anxiolytic and sedative effects of extracts and essential oil from Citrus aurantium L. Biol Pharm Bull., 25(12): 1629-33. Cates, M., Wells, B.G. and Thatcher, G.W. (1996). Anxiety Disorders. In: Textbook of Therapeutics: Drug and Disease Management, HerFindal, E.T. and Gourley, D.R. (editors), 6 th edition, Williams and Wilkins, Maryland, USA, pp. 1073-93. Chen, S.N., Li, W., Fabricant, D.S., Lu, Z.Z., Fong, H.H.S. and Farnsworth, N.R. (2002) Cimiracemates A-D, phenylpropanoid esters from the rhizomes of Cimicifuga racemosa. J. Nat. Prod., 61: 409-413. Chen, S.N., Li, W., Fabricant, D.S., Lu, Z.Z., Fong, H.H.S. and Farnsworth, N.R. (2002a). Cimiracemosides I-P, new 9,19-cyclolanostane triterpene glycosides from Cimicifuga racemosa. J. Nat. Prod., 65: 1391-1397. Cubukcu, B., Bray, D.H., Warhurst, D.C., Mericli, A.H., Ozhatay, N., Sariyar, G. (1990). In v~tro antimalarial activity of crude extracts and compounds from Artemisia

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9 Study of Eugenia jambolana and Momordica charantia in Respect to Safety and Antidiabetic Effect In vivo and In vitro YELE, SANTOSH 1 AND VEERANJANEYULU, A.l,*

ABSTRACT Diabetes mellitus is composed of a myriad of derangements on carbohydrate, lipid and protein metabolism, which are associated to an absolute or relative deficiency of insulin secretion / action. Out of a large number of herbal drugs in the Ayurvedic system of medicine of India, Eugenia jambolana of family Myrtaceae and Momordica charantia from cucurbitaceae (Jamun) is being widely used to treat diabetes by the traditional practitioners over many years. They are prepared as an aqueous or ethanolic extract, by infusion or as a juice of the raw plant. MC has a long history of use as a folklore hypoglycaemic agent where the plant extract has been referred to as vegetable insulin Eugenia jambolina (EJ) is well known plant with identified antidiabetic, laxative, anti-inflammatory and antimicrobial properties in traditional literature. In the present study the plants have been explored for the following parameters like safety evaluation, antidiabetic properties and in vitro antidiabetic properties, individually as well as in combination (1: 1) to find out additive of synergistic effect. Acute oral toxicity of water extract of Eugenia jambolana was evaluated in albino mice of both sexes. These results show that the water extract of Eugenia jambolana is toxicologically safe by oral administration. The effects of Eugenia jambolana bark extracts were studied with alloxan induced diabetes model for antidiabetic property. Albino Wistar rats weighing 150-200 g (N = 6) were fed E. jambolana bark extract and combination of EJ + MC (50:50) 500 mgt kg of b.w. for 14 days. In another investigation, we have studied the effects 1. Department of Pharmacology, School of Pharmacy and Tech. Management NMIMS

University, Mumbai - 400 056 Maharashtra, India.

* Corresponding author: E-mail: [email protected]

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ofaqueous fruit extract ofMe and aqueous bark extract ofEJ on the transport of d-glucose, I-tyrosine across rat-everted intestine in vitro. Everted intestinal sacs from rats were mounted in an organ bath containing Krebs-Henseleit bicarbonate buffer. Graded concentrations (1.5-12 mg/mL) of Me and EJ extract were incubated in the mucosal solution. The serosal appearance and mucosal disappearance ofd-glucose, and I-tyrosine were significantly inhibited with increasing graded concentrations of Me and EJ extracts. The aqueous extract of the plants was found to inhibit primarily the uptake of glucose in a dose-dependent manner. Uptake of tyrosine was affected at high substrate concentrations only. It is hypothesized that bioactive phytochemicals such as sap on ins in Me fruit extract and tannins from EJ inhibits the active transport of d-glucose, I-tyrosine across rat intestine It is revealed that Me and EJ can be a potential alternative drug therapy of postprandial hyperglycemia via inhibition of glucose uptake across the small intestine and could involve a washout of glucose from the blood stream. Key words: Acute toxicity, intestinal uptake, Eugenia jamb alana, Momordica charantia

INTRODUCTION Diabetes mellitus is a group of metabolic disorders characterized by hyperglycemia. These metabolic disorders include alterations in the carbohydrate, fat and protein metabolisms associated with absolute or relative deficiencies in insulin secretion and/or insulin action. Insulin is the hormone produced in the p-cells of pancreas which enables the cells to absorb glucose from the blood and also help in the utilization ofthe glucose in the cells by glycolysis, tricarboxylic acid cycle, hexose monophosphate shunt, glycogenesis etc. In diabetes body fails to produce insulin and excess glucose accumulates in the blood instead of being utilized or stored. The characteristic symptoms of diabetes are polyuria, polydypsia, polyphagia, pruritus and unexpected weight loss etc. Diabetes can be classified into two types: (a) type 1 diabetes (insulin-dependent diabetes mellitus or IDDM) and (b) type 2 diabetes (non-insulin dependent diabetes mellitus or NIDDM) (Guyton, 2002). Exercise and having a controlled diet are recommended for the treatment of both types of diabetes. In addition, insulin is used to treat cases of type 1 diabetes. Oral hypoglycemic agents (OHA) such as sulfonylureas, biguanidines, thiazolidinediones and a-glucosidase inhibitors are often used to treat cases of type 2 diabetes. When therapy with oral hypo-glycemic agents is ineffective, insulin also can be used to treat type 2 diabetes (Liu & Wang, 1996; Zhao, 1999). Management of diabetes without any side effects is still a challenge to the medical system. There is an increasing demand by patients to use the natural products with antidiabetic activity, because insulin and ORAs are having so many side effects. Medicinal plants continue to provide valuable

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therapeutic agents, both in modern medicine and in traditional system. The doubts about the efficacy and safety of the oral hypoglycemic agents have prompted a search for safer and more effective drugs in the treatment of diabetes (Reaven, 1983). A wide variety of the traditional herbal remedies are used by diabetic patients, especially in the third world countries (Lin, 1992; Ospinag & Serrano, 1995; Day, 1998; Gray & Flatt, 1998; Shimada et al., 1998) and may therefore, represent new avenues in the search for alternative hypoglycemic drugs. The folk medicine in India has described several kinds of ayurvedic herbs and plant crude drugs, belonging to various families to be concerned with the treatment of diabetes mellitus. Plant drugs are frequently considered to be less toxic and more free from side effects than synthetic ones (Pari & Umamaheswari, 2000). In the traditional system ofIndian medicine plant formulation and in several cases, combined extracts of plants are used as the drug of choice rather than individual. Presently several research laboratories are involved in isolating new herbal oral hypoglycemic agents or potent herbal formulation. The renounced OHA, Metformin which gained popularity in recent years got its shape from its ancestor, active ingredient Galegine or isoamylene guanidine from Galega officinalis (Cusi & DeFronzo, 1998). More than 100 medicinal plants are mentioned in the Indian system of medicines including folk medicines for the management of diabetes, which are effective either singly or in combinations. Many of these have shown promising effects (Kumari & Devi, 1993.). Various herbal formulations like D-400 (Mitra et al., 1996) and Trasina (Bhattacharya et al., 1997) are well known for their antidiabetic effects. While going through the previous works of different investigators, some variations in their experimental results have been observed. Incomplete extraction procedure, inadequate pharmacognosy and insensitive animal model may be the reasons for showing variable and even negative results. We have guarded against all these factors in our experiments and selected only two hypoglycaemic medicinal plants for our studies. Out of which one is Momordica charantia, traditionally and experimentally known for the antidiabetic activity (Grover et al., 2002) and another crude drug is Eugenia jambolana L. (Bark) less know part of plant and hardly explored for its use in diabetes as compared to its other parts like leaves, fruit and pulp (Nadakarni, 1954). Aim of present study is to confirm the ethnobotanical claim of antidiabetic property of Eugenia jambolana bark, and its use with momordica charantia. Folklore literature state that bark ofEJ and fruit powder ofMC exhibit the synergistic effect and control both blood glucose and body weight with short period of time as compared to individual drugs. Sahyadri hills row (Pune-Kolhapur region, Maharashtra, India), which lie geographically in the Western Ghats, are known for the rich heritage of the flora. A number of plants with known and unknown medicinal values

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are available here, which have to be explored for their use in the effective treatment of diabetes mellitus as described in the folk literature. But no systematic scientific studies were carried out on these plants by that mean.

MATERIALS AND METHODS Plant Material The bark of Eugenia jambolana and fruits of Momordica charantia were collected from local farm of Baramati (Maharashtra) and Pune region of Western Ghats of Maharashtra, respectively. Both the plant parts were authenticated by the scientists of BSI (Botanical Survey of India) Pune, Maharashtra. After collection, Bark of EJ was dried under shade and then washed out debris and adhered matter and again dried to remove moistness of bark. Bark was applied to disintegrator to make the fine powder. Same way fruits of Me were shade dried after proper removal of seeds and washing with water and powdered to coarse size.

Preparation of Extracts For entire experimentation cold macerated extract of both the plants were used. Powdered bark of EJ and fruits of MC were subjected for cold maceration up to 48 h. At the end of maceration syrupy liquid of both the drugs were obtained, to make it free flow powder extract were concentrated and dried under the reduced pressure with the help of rotary vacuum evaporator (Rotavap®). The yield of crude extracts from EJ and MC was 16.6% and 33.1% respectively. Extract of EJ was used individually and in combination with MC in the proportion of (EJ+MC-50:50). Alloxan was purchased from SD fine chemicals Company Inc., while all other chemicals used in this experiment were purchased from SD fine chemicals.

Acute Toxicity of EJ Bark Swiss albino mice were used for oral acute toxicity study and divided into four groups, each containing five animals. The bark extract was administered orally at doses of 1000, 3000, and 5000 mg/kg body weight. Distilled water was administered to control group. The animals were evaluated at 0.5,1,2,4,24 and 48 h, after administration to assess possible clinical or toxicological symptoms. Signs of toxicity and mortality were observed daily for 13 days. At the end of 14th day after the last observation the mice were killed and the liver, lungs, heart, spleen and kidneys were withdrawn, weighed and carefully examined macroscopically for any abnormal, pathological signs of toxicity.

Experimental Animals for Antidiabetic activity Male wistar rats weighing 180-200 g were obtained from the Haftkins' Research Institute Mumbai. The rats were housed at a regulated

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temperature (22°C) and humidity (55%) controlled room with a 12 h light: 12 h dark cycle. A water and standard pellet diet were available ad libitum throughout the experimental period.

Antidiabetic activity Effect of the crude extracts on serum glucose level in alloxan-induced diabetic rats Diabetes was induced by a single intravenous injection of 60 mg/kg of alloxan monohydrate (dissolved just before use in 0.9% NaCl) to overnight fasted rats (Silva et al., 2002 with modification). A serum glucose range of 400-550 mg/dl was used for the experiment. Animals in which the development of hyperglycemia was confirmed (around 90%), 72 h after the alloxan injection, were randomly allocated into four groups of five rats to each treatment: Group I, diabetic control received the saline solution Group II, non-diabetic received distilled water Group III, diabetic received the crude EJ extract 500 mg/kg b.w. Group IV, diabetic received the combined extracts EJ+MC (50:50) 500 mg/kg b.w Blood samples were collected from the retro-orbital plexus before the administration of extract or combination serum glucose levels were measured at zero time (before receiving the extract).

Effects of (MC+EJ) on transport of d-glucose and l-tyrosine across the intestinal sac - in vitro Adult male Swiss albino rats weighing 200-250 g maintained on commercial feed were used for this study. After overnight fasting, rats were killed by a severe blow on the head against a hard surface. The abdomen was opened by a midline incision. The whole of the small intestine was removed by cutting across the upper end of the duodenum and the lower end of the ileum and manually stripping the mesentery. The small intestine was washed out with normal saline solution (0.9% w/v NaCl) using a syringe equipped with blunt end. Intestinal segments (1062 cm) were then everted according to the method described by Wilson and Wiseman. After weighing, the empty sac was filled with 1 mL of Krebs- Henseleit bicarbonate buffer (KHB). The composition of the buffer was (mmll): NaHC0 3 25; NaCI 118; KCI 4.7; MgS0 4 1.2; NaH 2 P0 4 1.2; CaCl 2 1.2; and Na 2 EDTA 9.7 mg/l. Glucose (2 gil) was added to the medium just before the start of the appropriate experiments. The pH was maintained at 7.4. The sac was filled with a blunted-ended syringe and then slipped off the needle carefully and the loose ligature on the proximal end was tightened. After weighing, the

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distended sac was placed inside an organ bath containing 50 mL of the same incubation medium. The organ bath was surrounded by a water jacket maintained at 37-40°C and placed in metabolic shaker at a frequency of 100-110 shakes/min. The incubation medium was continuously being bubbled with a mixture of95% 02 and 5% CO 2 , At the end of the incubation period (30 min.), the sacs were removed from the organ bath, blotted by a standardized procedure and weighted again. The serosal fluid was drained through a small incision into a test tube. The emptied sac was shaken gently to remove the adhered fluid and the tissue was weighted again. The weight of the empty sac before and after the incubation did not differ significantly. The initial serosal volume was determined as the difference between the weights ofthe empty and filled everted sac prior to incubation. The final serosal volume was calculated by subtracting (after incubation) the weight of the empty sac from that of the filled sac (Mahomoodally et al., 2005, 2006). The aqueous EJ extract individually and with MC extracts (50:50) were incubated in the mucosal solution in the organ bath. The active transport of d-glucose and I-tyrosine were evaluated by measuring the increase in concentration of both compounds inside and outside the intestinal sacs after 30 min. of incubation. L-Tyrosine in the incubating buffer solution was determined as described by Lowry et al. (1951) with modifications. Glucose was measured using a commercially available glucose oxidase kit. The terms used for d-glucose and I-tyrosine transfer are mucosal glucose transfer, serosal glucose transfer and gut glucose uptake. Mucosal glucose transfer is the amount of glucose that disappeared from the mucosal fluid while serosal glucose transfer is the amount of glucose that entered the serosal fluid. Gut glucose uptake is the difference in glucose concentration between the mucosal and serosal fluid after incubation.

RESULTS AND DISCUSSION Acute Oral Toxicity Acute oral toxicity study of Eugenia jambolana extract showed that no mortality of mice occurred, at the doses of 1000, 3000, and 5000 mg/kg body weight given orally. This is an indication that the extract has negligible level of toxicity when administered orally. Besides, no signs of toxicity, such as convulsion, vomiting, diarrhoea, paralysis, breathing difficulties, bleeding, restless, irritation, and abnormal posture, were observed in Eugenia jambolana extract-treated mice. Thus, it can be suggested that Eugenia jambolana extract is virtually nontoxic. In conclusion, Eugenia jambolana extract was found to be fairly nontoxic when oral acute toxicity study in mice was performed.

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Effect of the crude extracts on serum glucose level in alloxan-induced diabetic rats Blood sugar level increased as expected in alloxan-injected animals, since alloxan causes a massive reduction in insulin release, by the destruction of the beta-cells of the islets of Langerhans and inducing hyperglycemia (Goldner et al., 1943). Oral administration of EJ (500 and mg/kg b.w.) and combined form of extract EJ+MC (500 mg/kg b.w.) resulted in a significant reduction in the blood glucose. In our study we have found that EJ decreases blood glucose in alloxan diabetic rats. In this context a number of other plants have also been observed to have hypoglycaemic effects. But when the results compared with individually action of EJ and its combination (EJ+MC) shows remarkably reduction in elevated blood glucose (Fig 1). The possible mechanism for hypoglycemic action may be potentiating the insulin effect of plasma by increasing either the pancreatic secretion of insulin from the ~-cells of islets of Langerhans or its release from bound insulin. As MC is suppose to known to posses insulin like compound and EJ posses tannins, triterpenes responsible for activity of reduction in plasma blood glucose. We have demonstrated that the folk medicinal plant EJ possesses a hypoglycemic effect. Further, active research is underway in our laboratory to elucidate the mechanisms of action of this medicinally important plant.

Effects of (Me + EJ) on transport of d-glucose and l-tyrosine across the intestinal sac - in vitro The effects of EJ crude aqueous extract individually and in combination with MC on d-glucose, I-tyrosine and transport (serosal appearance) across everted intestinal sacs of rat are summarized in (Tables 1 & 2). In order to find the lowest inhibitory concentration and any dose dependent relationship, graded concentrations of EJ and EJ+MC (from 1.5 to 12 mg/mL) were incubated with the intestinal segments in the mucosal solution. Data from the present investigation indicate that EJ inhibits significantly (p<0.05) the active transport of d-glucose, I-tyrosine. It was found that increasing graded concentrations of EJ+MC from 1.5 to 12.0 mg/mL in the mucosal solution decrease significantly the absorption as well as the transport of these compounds across rat intestine. The concentration of d-glucose accumulated or metabolized by the enterocytes (gut wall content) in EJ+MC was higher (pO.05) dose-dependent inhibition of d-glucose absorption and transport compared to 6, 3 or 1.5 mglmL EJ+MC.

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450 400

--+-- Diebetic control

~

350

__ NonDiabetic Control --+-- Diabetic + EJ 500mg/kg

__ Diabetic + (EJ+MC)

(11300

:g

500mg/kg

g 250 t; -g 200 o

iii 150 100 50

o

Day 0

Day 7

Day14 Days

Day 21

Day 28

Fig 1. Effect of the crude extracts on serum glucose level in alloxan·induced diabetic rats

MC fruit and EJ bark has been found to possess a wide array of bioactive phytochemicals and several classes of biologically active saponins have been isolated (Khanna et al., 1981; Murakami et al., 2001; Matsuur et al., 2002). Bioactive saponins, tannins constituents of natural medicines have been found to suppress the transfer of glucose from the stomach to the small intestine and inhibit glucose and fluid transport at the brush border membrane (Matsuda et al., 2001; Matsuur et al., 2002). Saponins constituents of herbs reduce the permeability barrier to Na+ at enterocytes, thus, discharging the electrochemical gradient and removing the driving force for sugar and fluid transport. Thus, the observed increased inhibitory effect of EJ+MC in the present study might be due to the bioactive compounds such as saponins present in MC fruit (Johnson et al., 1986). The results of the study reveals that EJ bark extract inhibits the uptake of d-glucose, I-tyrosine across rat everted intestinal sacs but when the same drug given in combination with MC shows greater effect by means of increased inhibition of glucose uptake. It is likely that MC fruit and EJ bark possess bioactive phytoconstituents which in combination capable of lowering postprandial blood glucose levels by inhibiting the uptake of glucose across the intestine and at same time increasing metabolism of glucose in cells. As most challenging factor in management of diabetes is to keep PBG as close as to the normal level. Present study supports to corroborate the folklore claim of use ofMC fruits with EJ bark in traditional medicines. In the conclusion, study can be a confirmatory document to claim combination of EJ+MC (50:50) potential alternative drug therapy of, postprandial hyperglycaemia.

Table 1. Effects of graded concentrations of aqueous EJ bark extract (1.5-12 mg/mL) on transport of d-glucose and I-tyrosine across rat everted intestinal sacs Concentration of EJ fruit extract in medium (mg/mL)

d-Glucose transport (MIg tissue wet weight) Mucosal Gut wall disappearance content

0 1.5 3

68.72

±

62.32 49.31

±

6 12

36.44 ± 2.83 40.22 ± 1.80#

±

1. 90 2.06 1.40#

Serosal appearance

15.10

±

1.21

54.10

±

24.37 29.82

±

1.73# 1.23#

40.05 29.63

±

±

24.84 ± 1.36 26.50 ± 0.93#

1.99

2.40# 0.03#,* 15.03 ± 0.86 14.90 ± 1.46# ±

Mucosal disappearance

I-Tyrosine transport (MIg tissue wet weight) Gut wall Serosal appearance content

16.08

±

0.76

1.39

±

0.14

12.91

±

1.42

15.01 12.82

±

1.41 0.83#

1.89 2.07

±

0.30# 0.30#

11.82 9.05

±

0.64 0.56#,*

±

10.03 ± 1.48 10.88 ± 0.68#

±

2.23 ± 0.24 2.09 ± 0.28#

±

8.70 ± 0.66 8.23 ± 0.55#

The results are expressed as means ± S.E.M of seven observations in each group, # p<0.05 from the control without EJ extract added to the mucosal solution. * p<0.05 from those values immediately above (between the different EJ concentrations). Table 2. Effects of graded concentrations of extract MC + EJ (50:50) (1.5-12 mg/mL) on transport of d-glucose and I-tyrosine across rat everted intestinal sacs d-Glucose transport (MIg tissue wet weight) Mucosal Gut wall disappearance content

Serosal appearance

Mucosal disappearance

79.56

±

1.99

13.13

±

2.02

66.43

±

2.12

16.30 ± 0.86

1.32

±

44.86 25.23

±

2.51#

±

±

25.57 29.10 22.22

1.73# 1.18#

41.95

2.08 1.29# 2.58#

±

3 6

70.61 54.33

±

1.36#

19.27

±

0.79# 0.93#

12.82 ± 1.24 10.03 ± 0.54# 09.82 ± 1.27#

1.66 ± 0.30# 1.87 ± 0.41# 2.53 ± 0.33#

1.37 0.42 08.16 ± 0.35# 07.29 ± 0.81#

12

51.03

±

1.87#

26.93

±

0.61#

24.10

±

2.09#

09.09 ± 0.84#

2.00 ± 0.20#

7.09 ± 0.28#

Concentration of EJ fruit extract in medium (mg/mL) 0 1.5

±

±

I-Tyrosine transport (MIg tissue wet weight) Gut wall Serosal appearance content ±

0.22

14.98

±

11.16

±

The results are expressed as means ± S.E.M of seven observations in each group, # p<0.05 from the control without EJ extract added to the mucosal solution. * p<0.05 from those values immediately above (between the different EJ concentrations).

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ACKNOWLEDGEMENTS Authors are indebted to the Prof. R.S. Gaud. Dean, School of Pharmacy and Technology Management, NMIMS University, for provision of all facilities to complete the research work. We are also grateful to Prof. S.B. Gokhale, Principal, SVKM's College of Diploma Pharmacy and Dr. Meena C. HOD Clinical Pharmacology for technical assistance.

REFERENCES Bhattacharya, S.K, Satyan, KS. and Chakrabrati, A. (1997). Effect of Trasina, an Ayurvedic herbal formulation, on pancreatic islet superoxide dismutase activity in hyperglycaemic rats. Ind. J. Exp. Bioi., 35: 297-299. Clarke, E.G.C. and Clarke, M.L. (1977). Veterinary toxicology, 268-277. London: Cassel and Collier Macmilan. Cusi, K and DeFronzo, RA. (1998). Metformin: a review of its metabolic effects. Diabetes Reviews, 6(2): 89-13l. Day, C. (1998). Traditional plant treatments for diabetes mellitus: pharmaceutical foods. Br. J. Nutr., 80: 5-6. Goldner, M. and Gomori, G. (1943). Alloxan induced diabetes. J Endocrinol., 33: 297-9. Gray, A.M. and Flatt, P.R (1998). Actions of traditional antidiabetic plant, Agrimony eupatoria (agrimony): effect on hyperglycemia, cellular glucose metabolism and insulin secretion. Br. J. Nutr., 80: 109-114. Grover J.K, Yadav S. and Vats V. (2002), Medicinal plants of India with anti-diabetic potential. Journal of Ethnopharmacology, 81: 81-100 Johnson, I.T., Jennifer, M., Gee, KP., Caroyn, C. and Fenwick, G.R (1986). Influence of saponins on gut permeability and active nutrient transport in vitro. Journal of Nutrition, 116: 2270-2277. Khanna, P., Jain, S.C., Panagariya, A. and Dixit, V.P. (1981). Hypoglycaemic activity of polypeptide-p from a plant source. Journal Natural Products, 44: 648-655. Kumari, KS. and Devi, KS. (1993). Effect of indigenous drugs on glucuronoglycan metabolism in diabetic hypertensive rabbits. Ind. J. Exp. Biol., 31: 595-599. Lin, C.C. (1992). Crude drugs for the treatment of diabetes mellitus in Taiwan. Am. J. Chin. Med., 20(3-4): 269-279. Liu, RY. and Wang, G.Q. (1996). A survey on drugs synthesized for antidiabetes. Journal of Shenyang Pharmaceutical Unwersity, 13: 148-153. Lowry, O.H., Rosebrough, N.J., FaIT, A.L. and Randall, RJ. (1951). Protein measurement with the folin phenol reagent. J. Bioi. Chem., 193: 269-275. Lowry, O.H., Rosenbrough, N.J., FaIT, A.L. and Randall, RJ. (1951). Protein measurements with the Folin phenol reagent. Journal of Biological Chemistry, 193: 265-275. Matsuda, H., Murakami, T., Li, Y., Matsumura, N., Yam ahara, J. and Yoshikawa, M. (1998). Antidiabetic principle of natural medicines. III. Structure-related inhibitory activity and action mode of oleanolic acid glycosides on hypoglycemic activity. Chemical Pharmaceutical Bulletin, 46: 1399-1403. Matsuur, H., Asakawa, C., Kurimoto, M. and Mizutani, J. (2002). Alpha-glucosidase inhibitor from the seeds of balsam pear (Momord!ca charantia) and the fruit bodies of Grifola frondosa. Bioscience Biotechnology Biochemistry, 66: 1576-578. Mitra, S.K, Gopumadhavan, S., Muralidhar, T.S., Anturlikar, S.D. and Sujatha, M.B. (1996). Effect of a herbomineral preparation D-400 in streptozotoc in induced diabetic rats. J. Ethnopharmacoi., 54: 41-46. Murakami, T., Emoto, A., Matsuda, H. and Yoshikawa, M. (2001). Medicinal foodstuffs. XXI. Structures of new cucurbitane-type triterpene glycosides, goyaglycosides-a, -b, -c, -d, -e, f, -g and -h, and new oleanane-type triterpene saponins, goyaglycosides I, II and III, from the fresh fruit of Japanese Momordica charantia L. Chemical Pharmaceutical Bulletin, 49: 54-63.

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Nadkarni, A.K. (1954). Indian Materia Medica, Popular Prakashan, Bombay, 1221. Ospinag, L.F. and Serrano, RP. (1995). Plants used as antidiabetics in Colombian folk medicine. Rev. Colomb. Clenc. Quimico·Farm., 23: 81-94. Reaven, G.M. (1983). Effect of age and diet on insulin secretion and insulin action in the rat. Am. J. Med., 74: 69. Sharma, R and Misra, A. (1993), Autoimmunity and gestational diabetes. The National Medlcal Journal of Indw, 6: 272-273. Shimada, H., Kawamori, S. and Kawahara, Y. (1998). Effect of an aqueous extracts of Salacia reticulata, a useful plant in Srilanka, on postprandial hyperglycemia in rats and human. Nippon Eiyo Shakuryo Gakkaiski., 51: 279-287. Silva, F.RM.B., Szpoganicz, B., Pizzolati, M.G., Willrich, M.A.V. and Souza, E. (2002). Acute effect of Bauhinia forficata on serum glucose levels in normal and alloxan-induced diabetic rats. Journal of Ethnopharmacology, 83: 33-37. Trinder, P. (1976). In: Varley, H., Gowenlock, A.H. and Bell, M. (Eds.). Practical Clinical Biochemistry. William Heinemann Medicinal Books Ltd., London, p. 389 Zhao, S.Y. (1999). Drugs to treat Diabetes. Guowai Yiyao-Hechengyao, Shenghuayao, Zhiji, 20: 130-135.

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10 Larvicidal and Antimicrobial Activities of Seeds of Annona cornifolia A. St. -Hil. (Annonaceae) LUCIANA ALVES R.S. LIMA\ MARIAAMELIAD. BOAVENTURA\ JACQUELINE A. TAKAHASHI l AND LUCIA P.S. PIMENTA l

*

ABSTRACT The hexane and ethanol extracts from Annona cornifolia A. St. -Hil seeds showed expressive activity in Artemia salina (brine shrimp) and antimicrobial tests. A fraction from hexane extract rich in fatty acids and the corresponding fatty acid methyl esters fraction showed antibacterial and antifungal activities. Methyl esters were prepared from saponifiable fraction of hexane extract with methanol/sulfuric acid and were identified by GC and GC-MS. A fraction obtained from chloroform extract, positive for acetogenins, was extremely active against Candida albicans. Key words : Annona cornifolia, Annonaceae, antimicrobial activity, fatty acid methyl esters, larvicidal activity

INTRODUCTION Plants from Annonaceae family are very important sources of edible fruits and they are used in folk medicine as antitumor, parasiticide, insecticide, antifever, antidysentery, antiedema (Cepleanu et al., 1993), anti-influenza, anti-insomnia (Wang et al., 2002) and antidiarrhoea agents (Correa et al., 1984; Leboeuf et al., 1982). Annona is far the greater genus in this family and species such as A. crassiflora, A. squamosa and A. muricata have a wide use in traditional medicine against diabetes, malaria and infections (Sousa et al., 1991). These activities, from the phytochemical viewpoint, were initially associated only with the isolation of alkaloids, very common 1. Departamento de Quimica, Instituto de Ciencias Exatas, UFMG, Av. Antonio Carlos 6627, Belo Horizonte, MG, CEP.: 31270-901, Brazil. * Corresponding author: E-mail: [email protected]

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phytochemical constituents of this family. Nowadays, several activities associated to other classes of natural compounds isolated from this family, such as annonaceous acetogenins, have been reported (Alali et al., 1999). Annona cornifolia A. St. -Hil. is native from southeast of Brazil and its green fruits are popularly used to heal chronic ulcers (Correa, 1984). The only phytochemical study of this species, to our knowledge, has been done by our group and the isolation and characterization of three acetogenin were described (Santos et al., 2006; Santos et al., 2007).

Since most of the activities related to Annonaceae plants, including Annona species, concern to their anti-infectious potential, A. cornifolia was screened by larvicidal (Brine Shrimp Lethality test) and antibacterial! antifungal protocols.

MATERIALS AND METHODS General

IR spectra were recorded on a ShimadzulIR-408 spectrophotometer. Fatty acids methyl esters analysis was carried out with a Varian CP-3380 Gas Chromatograph using hydrogen as the carrier gas. A GE Science BPI column (12 m x 0.25 mm, 100% dimetylpolysiloxane, 0.25 mm) was used. The temperature of the GC oven was held at 150°C and programmed to 240°C with increase of lOoC/min; injection temperature was 250°C and detector, temperature 260°C. Split ratio was 1/80. Analysis GC-MS were performed in a Hewlett Packard 5890 Serie II Chromatograph coupled in a Hewlett Packard 5989A mass spectrometer, using electron ionization. Oven flow was 2 mUmin and mass range was 40-400 m/z. The fatty acids methyl esters were identified by comparison of their retention times with those of standards and by using the Wiley 138 Library database search. Plant Collection, Identification and Extract Preparation

Fruits of Annona cornifolia A. St. -Hil. were collected in Curvelo city region, Minas Gerais State, Brazil, from January to March 1998. The species was identified by Dr. R. Mello-Silva and a voucher specimen (BHCB 68114) was deposited at the Instituto de Ciencias Biologicas Herbarium, Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil. The fruits were open, the seeds removed, washed, dried at 40°C, powdered (850.0 g) and extracted exhaustively with hexane to give extract F01 (79.5 g) and then with ethanol to give extract F02 (120.0 g), after solvent removal. The latter was dissolved in methanol!water (7:3) and extracted with chloroform to give F03 (7.3 g). Hexane extract (F01) was fractionated by column chromatography on silica gel using gradient systems of n-hexane/CH 2 Cl/AcOEtlCH3 0H in increasing polarities; a fraction rich in fatty acids (F01-1, 53.2 g) was isolated. Part of this fraction (500 mg)

Larvicidal and Antimicrobial Activities of Seeds of Annona cornifolia

233

was refluxed with 1.0 mollL methanolic sodium hydroxide solution for 30 min. and then extracted with ethyl ether. The aqueous phase was acidified with 1.0 mollL HCI solution and extracted with ethyl ether. The organic phase was dissolved in hexane and then refluxed with H 2S0 4 methanolic solution 2% v/v for 1 h. 370 mg of a mixture of fatty acid methyl esters (F01-1Me) were obtained after extraction and solvent elimination. Part of chloroform fraction (F03, 1.0 g) was submitted to medium pressure liquid chromatography using hexane/toluenlAcOEtlEtOHlMeOH in increasing polarities, to furnish several fractions, that were tested for acetogenins by TLC. Acetogenins containing fractions were expected to show pink color by Kedde's reagent and orange color by Dragendorffs reagent. Infrared profile were also obtained. Only fraction F03-7 was positive for acetogenins. Afterwards acetogenins were isolated from this fraction and were related elsewhere (Santos et al., 2006; Santos et al., 2007; Lima et al., 2008).

Larvicidal Bioassay The methodology used was described by Pimenta et al. (2003) and Stefanello et al. (2006). Artemia salina encysted eggs (10 mg) were incubated in 100 mL of seawater under artificial light at 28°C and pH 7-8. After incubation for 24 h, nauplii were collected with a Pasteur pipette and kept for additional 24 h under the same conditions to reach the metanauplii stage. The samples (triplicate) to be assayed were dissolved in dimethylsulfoxide (DMSO) (2 mg/400 ilL or 2 mg/1000 ilL) and diluted serially (10, 20, 30, 50 IlU5 mL) in seawater. About 10-20 nauplii were added to each set of tubes containing the samples. Controls with 50 ilL of DMSO in seawater were included in each experiment. Lapachol dissolved in DMSO was used as positive control. 24 h later, the number of survivors was counted, recorded and the lethal concentration 50% (LC 5o value) and 95% confidence intervals were calculated by probit analysis (Finney, 1974).

Antimicrobial Bioassay The methodology used was described by Miguel et al. (1996); Bisagno et al. (2000); Almeida et al. (2006) and Smania et al. (2007). Staphylococcus aureus (ATCC 25985), Streptococcus pyogenes (ATCC 19615), both gram positive, Salmonella typhimurium (ATCC 13311), gram negative and the yeast Candida albicans were used for this test. The minimum inhibitory concentration (MIC) was evaluated by the macro dilution test using standard inoculums of 10-5 CFU mL-l. Serial dilutions of the test compounds, previously dissolved in DMSO, were prepared to final concentrations of 550.00, 275.00, 137.50, 68.75, 34.38, 17.18, 8.59, 4.30, 2.15 and 1.07 llg/mL. To each tube were added 100 ilL of a 24 h old inoculum. The MIC, defined as the lowest concentration of the test compound, which inhibits the visible growth after 18 h, was determined visually after

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incubation for 18 h at 37°C. Tests using DMSO as negative control and chloramphenicol and ketoconazol as positive controls were carried out in paralleL All tests were performed in duplicate with full agreement between both results.

RESULTS AND DISCUSSION Phytochemical analysis of extracts and fractions obtained from A. cornifolia seeds led to the identification of a fraction positive for acetogenins (F03-7), obtained from chloroform extract (F03). Both F03 and F03-7, as well as F01 (hexane extract), F01-1 (fatty acids fraction), F01-1Me (fatty acid methyl esters fraction) and F02 (ethanol extract) were tested for cytotoxicity by brine shrimp lethality (A. salina) test. The results obtained from this bioassay are shown in Table 1. LD50 values, with 95% confidence range, in IJ,gimL, were determined using probits method (Finney, 1974). Hexane extract (F01), fraction rich in fatty acid (F01-l), and fatty acid methyl ester fraction (F01-1Me) showed good cytotoxic activity (LD 50 = 10.00, 9.60 and 8.77 IJ,g/mL, respectively), even higher than the activity presented by the standard, lapachol LD50 = 70 mgimL (61 < LD50 < 81). Fraction F01-1Me was analysed by gas chromatography and the following fatty acid methyl esters were identified: miristate (C-14); palmitate (C-16); stearate (C-18); oleate (C-18, ~9); linoleate (C-18, ~9,12) and linolenate (C-18, ~9,12,15), in 0.17, 16.92, 5.56, 51.46, 19.12 and 0.81%, respectively. Table 1. Larvicidal (brine shrimp lethality bioassay) and antimicrobial (minimum inhibitory concentration, MIG) activities for extracts and fractions from seeds of Annona cornifolia Extracts and fractions

FOl F02 F01-1 F01-1Me F03 F03-7 Lapachol b Ghloranphenicol Ketoconazol d

Larvicidal Minimum inhibitory concentration (mg/mL) (MIe) activity Salmonella StaphyloccoccusStreptococcus Candida aureus pyogenes albicans typhimurium LDso a(Wl/mL) 10.00 0.20 9.60 8.77 0.13 0.16 68.09 C

137.50 137.50 275.00 550.00 137.50

68.75 137.50 137.50 275.00 137.50

137.50 137.50 137.50 275.00 137.50

8.40

33.60

4.20

34.38 68.75 68.75 nd 2.15

40.60

a Dosis that kills 50% ofthe individuals of a population; b control used in the brine shrimp larvicidal bioassay; C control used in the MIG test for S. aureus, S. pyogenes, Salmonella typhimurium; d control used in the MIG test for C. albicans; nd = not detected.

Larvicidal and Antimicrobial Activities of Seeds of Annona cornifolia

235

The saponifiable part of the fraction F01-1 showed 6 fatty acids identified as methyl ester. The total of unsaturated fatty acids methyl esters found in F01-1Me was 71.39% (Table 2). Polyunsaturated fatty acids, when screened by the brine shrimp A. salina test, are reported to have higher toxicity compared to satured fatty acids (lkawa, 2004). According to this report, the toxic activity of such fatty acids hypothetically arises from the corresponding oxidation products, derived from photooxidation or metabolic processes. Table 2. Fatty acid composition ofthe FOl-l hexane fraction of A. cornifolia seeds in its methyl ester form Compund ~ethylmyristate ~ethyl

palmitate ~ethyl stearate ~ethyl oleate ~ethyllinoleate ~ethyllinolenate

Relative percentage 0.17 16.92 5.56 51.46 19.12 0.81

F02, F03 and F03-7 were highly active (LD 50 = 0.20 llg/mL, 0.13 and 0.161lg/mL, respectively), in brine shrimp CA. salina) test, presenting results compatible with literature values found for mixtures of acetogenins (Pimenta et al., 2003). All fractions were also tested for their antimicrobial activity. Minimum inhibitory concentration (MIC) was determined by macrodilution method for ten test concentrations ranging from 550 to 1.07 /lWmL. MIC values were determined after incubation at 37°C, for 14 h. All extracts and fractions were active (Table 1). F03-7 was not tested, due the little amount available. F01, constituted by a mixture of fatty acids, showed activity, against S. aureus, close to the control value. It is already known that even traces of certain long chain fatty acids inhibit microorganisms growth, specially Gram-positive bacteria, by affecting cell permeability and transport of nutrients (Wang & Johnson, 1992). Fatty acid monoesters of glycerol have strong bacteriostatic activity against B. subtilis and S. aureus (Kato & Shibasaki, 1975). According to Kabara (1987), esterification of polyhydroxyl compounds, as glycerol, with fatty acids generally leads to more active derivatives, whereas the same reaction with monohydroxyl compound cause deactivation of the fatty acid. In agreement with this information, esterified fraction (F01-1Me) showed less activity than the original fraction containing the fatty acids. F03, rich in acetogenins, showed good activity against Salmonella typhimurium, Staphylococcus aureus and Streptococcus pyogenes. Acetogenins isolated from Uvaria hookeri and Uvaria narum are reported to possess antibacterial and antifungal activities (Padmaja et al., 1993).

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The present study shows that traditional use of A. cornifolia fruits, such as against ulcers, can be associated to the cytotoxic and antibacterial activities found in their extracts. A. cornifolia seems to be extremely interesting as source of novel antifungal agents. Both hexane and ethanol extracts (F01 and F02) were highly active against Candida albicans, F01 being even more active than the control used (ketoconazol). F03, the chloroform fraction obtained from F02, presented a MIC = 2.15 ~g/mL, being almost 20 times more active than the control. From the fraction F03 five acetogenins were isolated that have showed great activity in Brine Shrimp Lethality test (Santos et al., 2006; Santos et al., 2007; Pimenta et al., 2008).

AKNOWLEDGEMENTS The authors thank Conselho Nacional de Desenvolvimento Cientifico e Tecnol6gico (CNPq) for LARSL, MADB and JAT grants. The authors also thank FAPEMIG for financial support (grant CDS-853106).

REFERENCES Alali, F.Q., Liu X.-X. and McLaughlin, J.L. (1999). Annonaceous acetogenins: recent progress. J Nat Prod., 62: 504-540. Almeida, J.RG.S., Filho RN.S., Nunes, X.P., Dias, C.S., Pereira, F.O. and Lima, E.O. (2006). Antimicrobial activity of the essential oil of Bowdichia virgilioides Kunt. Rev Bras Farmacogn., 16: 638-641. Bisagno, G., Sanogo, R, Marino, A., D'Angelo, V., Germano, M.P., De Pasquale, R and Pizza C. (2000). Antimicrobial activity of Mitracarpus scaber extract and isolated constituents. Letters in Appl Microbiol., 30: 105-108. Cepleanu, F., Ohtani, K., Hamburger, M., Gupta, M.P., Solis, P. and Hostettmann, K. (1993). Novel acetogenins from leaves of A. purpurea. Helv Chim Acta, 76: 1379-1388. Correa, M.P. (1984). Dicionario de plantas uteis do Brasil e de plantas ex6ticas cultivadas. Rio de Janeiro: Imprensa Nacional, v. 1, pp. 151-162. Finney, D.J. (1974). Probit analysis, a statical treatment of sigmoid response curve. 3 ed. Cambrige: University Press, pp. 76-80. Ikawa, M. (2004). Algal polyunsaturated fatty acids and effects on plankton ecology and other organisms. UNH Center for Freshwater Biology Research, 6: 17-44. Kabara, J.J. (1987). Fatty acids esters as antimicrobiallinseticidal agents. In: ACS Symposium Series 325 (Ecol. Metab. Plant Lipids): 220-238. Kato, N. and Shibasaki, 1. (1975). Comparison of antimicrobial activities of fatty acids and their esters. J. Ferment Technol., 53: 793-801. Leboeuf, M., Cave A., Braumik, P.K., Mukherjee, B.E. and Mukherjee, R (1982). The phytochemistry of the annonaceae. Phytochemistry, 21: 2783-2813. Miguel, O.G., Lima, E.O., Morais, V.M.F., Gomes, S.T.A., Monache, F.D., Bella-Cruz, A., Bella-Cruz, RC. and Cechinel, F.v. (1996). Antimicrobial activity of constituents isolated from Lichnophora sal~cifolia (Asteraceae). Phytoter Res., 10: 694-696. Padmaja, D., Thankamany, V. and Hisham, A. (1993). Antibacterial, antifungal and antihelmintic activities of root barks of Uvaria hookeri and Uvaria narum. J Ethnopharmacol, 40: 181-186. Pimenta, L.P.S., Pinto, GB, Takahashi, J.A., Silva, L.G.F. and Boaventura, M.A.D. (2003). Biological screening of annonaceous Brazilian medicinal using Artemia salina (brine shrimp test). Phytomedicine, 10: 209-212.

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Pimenta, L.P.S., Lima, L.A.R. dos S. and Boaventura, M.A.D. (2008). Submitted for publication. Santos, L.A.R., Boaventura, M.A.D. and Pimenta, L.P.S. (2006). Cornifolin, a new bistetrahydrofuran annonaceous acetogenin from Annona cornifolia. Biochem System Ecol., 34: 78-82. Santos, L.A.R., Boaventura, M.A.D. and Pimenta, L.P.S. (2007). Acetogeninas de Anonaceas Bioativas isoladas das sementes de Annona cornifolia A. St.-Hill. Rev. Bras. PI. Med., 9: 48-5l. Smania, E.F.A., Monache, F.D., Yunes, R.A., Paulert, R. and Junior, A.S. (2007). Antimicrobial activity of methyl australate from Ganoderma australe. Rev Bras Farmacogn., 17: 14-16. Sousa, M.P., Matos, M.E.O. and Matos, F.J.A. (1991). Constituintes quimicos de plantas medicinais brasileiras. Fortaleza: Impr. UniversitarialUFC. Stefanello, M.E.A., Salvador, M.J., Ito, LY. and Macari, P.A.T. (2006). Avalia~i'i.o da atividade antimicrobiana e citot6xica de extratos de Gochnatia polymorpha ssp floccosa. Rev Bras Farmacogn., 16: 525-530. Wang, L.L. and Johnson, E.A. (1992). Inhibition of Listeria monocytogenes by fatty acids and monoglycerides. Appl Environ Microbwl., 58: 624-629. Wang, L.Q., Min, B.-S, Li, Y., Nakamura, N., Qin, G.-W., Li, C.-J. and Hattori, M. (2002). Annonaceous acetogenins from the leaves of Annona montana. Bioorg Med Chem., 10: 561-565.

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11 Antidepressant Activity of the Extracts of Three Hypericum species Native to China DEPO YANG!, JrE BAI l ,2, ZHEN Lr!, GEORGE

Q L r3 AND DONGMEr WANG l ,*

ABSTRACT

The objective of this study was to evaluate three Chinese species ofHypericum genus (Hypericaceae): Hypericum erectum subsp. longisepalum, H. hubeiense and H. seniavinii, as the new sources of antidepressant phytomedicine. The hydroalcoholic extracts of the three Hypericum plants were evaluated for their antidepressant effects by forced swimming test (FST) and tail suspension test (TST) in mice. The mechanisms of action were also investigated by 5-HT-induced head twitches, potentiation of NA toxicity, and elevated plus-maze in mice. All the three extracts (at the doses of250 or 500 mg/kg, or both) significantly shortened the immobility time in FST and TST. These extracts potentiated the head twitches induced by 5-HT, and H. hubeiense extract (at the dose of 500 mg/kg) also showed an increase of noradrenaline toxicity. In addition, H. erectum and H. hubeiense extracts showed anxiolytic action in the elevated plus-maze test. The results suggest that these three Hypericum extracts possess antidepressant activity, which may be mediated by serotonergic system, and noradrenergic system may also be involved for H. hubeiense. The three Chinese Hypericum species possess activity similar to H. perforatum and have the potential to be used as new sources of antidepressant products. Key words : Antidepressant, forced swimming test, hypericum, tail suspension test 1. School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510 080, P. R.

China. 2. Chemistry Department, Tsinghua University, Beijing 100 084, P.R. China. 3. Herbal Medicines Research and Education Centre, Faculty of Pharmacy, The University of Sydney, NSW 2006, Australia. * Corresponding author : E-mail: lsswdm®mail.sysu.edu.cn.

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INTRODUCTION Hypericum perforatum L. (St. John's wort) is herbaceous plant used for the treatment ofthe mild-to-moderate depression (Linde et al., 1996; Kasper et al., 2001). It is used in traditional Chinese medicine for the treatment of burns, bruises, swelling, inflammation, and skin diseases (Wu et al., 2002). It has been demonstrated that hypericins, hyperforin and flavonoids are the main active components (Jiang et al., 2004; Sanchez-Mateo et al., 2005). Recent reports have raised the concerns on its interference effect on the metabolism of some pharmaceutical drugs which may limit its future application for some patients (Jiang et al., 2004). Therefore, there is a need to improve the safety profile of St. John's wort and extracts from other species of Hypericum which may offer the alternative (Sanchez-Mateo et al., 2005). The current study aims to identify new source of Hypericum of antidepressant activity from Chinese Hypericum species. There are over 60 Hypericum species distributing in China, but only a few of them have been studied for their chemical constituents and pharmacological activities (Wu et al., 2002; Fu et al., 2006). Recently new species of Hypericum were collected and identified by our laboratory (Wu et al., 2004). In the current study, we reported the antidepressant activity and the possible mechanisms of three Chinese Hypericum: Hypericum erectum subsp. longisepalum L. H. Wu (H. erectum), H. hubeiense L. H. Wu et D. P. Yang, and H. seniavinii Maxim, using five well established animal models.

MATERIALS AND METHODS Plant Materials The aerial parts of H. erectum, H. hubeiense and H. seniavinii were collected in Badong (Hubei Province, China) during the flowering period in August 2001, and dried at room temperature avoiding direct sunlight. The voucher specimens (No. 01020, No. 01035, No. 01034, respectively) were deposited at Biological Museum of Sun Yat-sen University, Guangzhou, China. The aerial parts of these plant samples were powdered for extraction. The hydroalcoholic extracts were obtained by maceration of the powdered samples with 6-fold (w/v) amount of 90% aqueous ethanol for overnight at room temperature, and this procedure was repeated twice. The extracts were filtered and dried under reduced pressure below 45°C. The contents of hypericins (hypericin and pseudohypericin) were determined by HPLC and the total flavonoids determined by UV using published methods (Zheng et al., 2003).

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Drugs The following drugs were used as control in the study: imipramine hydrochloride (Jiufu, China), amitriptyline hydrochloride (Shizhou, China), 5-hydroxytryptophan (Acros, USA), N-methyl-N-propargyl-benzylamine (Pargyline) (Acros, USA), norepinephrine (Changji, China), and diazepam (Siyao, China).

Animals and Drug Administration NIH male mice (18-22 g) were provided by the Medical Animal Center, Guangzhou University of Chinese Medicine (China). The naive animals were kept in a 12 h light/dark cycle (light on started at 07:00) at ambient temperature of 25 ± 1°C. Plant extracts and control drugs were suspended in a 0.5% carboxymethyl cellulose aqueous solution (vehicle) and adjusted to various concentrations so that the final volume given orally was 15 mU kg, and the final dosage in mice were 250, and 500 mglkg body weight. They were given orally by gavage 1 h before the tests. Control animals received vehicle under the same conditions. Behavioral observations took place between 8:00 and 15:00 o'clock. All studies were performed in accordance with the Experimental Animal Management Regulations of National Science and Technology Commission, China (November 14th 1988, Article No.2). In each experimental paradigm, the observers were blind of the treatment protocol. Imipramine and amitriptyline (20 mg/kg) were given by intraperitoneal route (i.p.) 10 min before FST, and TST respectively. In the elevated plusmaze test, diazepam (3 mglkg) or vehicle was given orally 1 h before the tests were performed. Experiments were conducted between 8:00-15:00 o'clock. The mice were used just for one time. Each group of behavioral experiments consisted of 12 mice, unless otherwise stated.

Preliminary Acute Toxicity Limit test was performed to observe the acute toxicity of the three Hypericum extracts. There were six mice in each group. Animals received decreasing doses of H. erectum, H. hubeiense and H. seniavinii extracts with the initial dose of 5 glkg (p.o.), and were placed in empty cages to observe signs of toxicity. Observation was carried out until 7 days after administration.

Forced Swimming Test (FST) in Mice The forced swimming test was performed according to the methods described by Porsolt et al. (1977). Mice were individually placed in a cylindrical glass swim vessel (25 x 12 cm i.d.) filled with 12 cm of water maintaining at 23-25°C. The total immobility time was observed during the last 4 min of a single 6 min test session by unaided eyes of a trained observer, who was blind to the experimental conditions. Mice were

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considered immobile when they showed no further attempts to escape except the necessary movements to keep their heads above the water.

Tail Suspension Test (TST) in Mice After 1 h following withdrawal, mice were individually taken out from the vivarium to an adjoining room and immediately suspended by the tail to a horizontal ring-stand bar (The distance from the Table top was 60 cm) using adhesive tape affixed 1 cm from the tip of the tail. The testing room was brightly lit. The mouse was 15 cm away from the nearest object and both acoustically and visually isolated. The cumulative period of immobility during an observation period of 6 min was recorded by a video camera. The video tapes were scored by a trained observer who was blind to the experimental conditions. Mice were considered immobile only when they hung passively and were completely motionless (Steru et al., 1985).

5-HT-induced Head Switches in Mice Test drugs were orally administered 30 min before 100 mg/kg i.p. pargyline, and then 1 h later, i.p. 20 mg/kg 5-HT. Mter administration, the mice were placed in empty cages and the number of head twitches was counted during 15-35 min after 5-HT injection (Goodwin et al., 1984).

Potentiation ofNA Toxicity in Mice Eight groups of mice, ten in each group, were injected NA (3 mglkg) subcutaneously 60 min after being administrated the extracts (250 and 500 mg/kg, p.o.), vehicle (p.o.), and amitriptyline (20 mg/kg, p.o.), respectively. The number of dead mice was counted 48 h after the administration of test drug (Alpermann et al., 1992).

Elevated Plus-Maze in Mice The mouse plus-maze consisted of two opposite open arms (30 x 5 cm) crossed with two closed arms of the same dimension with 15 cm height. The arms were connected with a central square (5 x 5 cm) elevated 45 cm above floor level. The animal was placed individually in the center ofthe maze facing a closed arm. The number of entries into open and closed arms, and the time spent in each arm during the next 5 min were evaluated by an observer inside the room (Willner et al., 1984; Lister et al., 1987). During the experiment, when each test of 5 min was completed, the maze was carefully cleaned with 20% ethanol solution and then thoroughly dried.

Statistical Analysis Data analysis was performed by one-way analysis of variance (ANOVA) with the Dunnett post-hoc test for multiple comparisons by SPSS 10.0 software. All data were expressed as means ± S.E.M. and the level of statistical significance was set at p<0.05.

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243

RESULTS Preliminary Acute Toxicity In limit test, H. hubeiense extract did not cause any toxicity manifestations until a dose of 5 g/kg (p.o.), while H. erectum and H. seniavinii extracts induced mortality at lower doses. H. seniavinii and H. erectum extracts showed no toxicity with doses up to 3.5 g/kg, and 1.75 g/kg, respectively. It was observed that the body weight of the mice treated with H. seniavinii extract was significantly lower than those of control group 3 days after administration at a dose of 3.5 g/kg, and then recovered to normal body weight 7 days later. No body weight reduction was observed in H. erectum or H. hubeiense treated groups.

Behavioral Despair Test The activities of the three Hypericum extracts on the forced swimming test are shown in Fig 1. The tricyclic antidepressant imipramine (20 mg! kg) reduced the immobility time significantly (69.4 ± 8.5 seconds from control 105.9 ± 19.2 seconds, p<0.05). At 250 and 500 mg/kg, both H. hubeiense and H. erectum extracts, shortened the immobility time of mice significantly in FST (p<0.05 or p
~ (/J

~

~

r:J 250

140 120 100 80

mg/kg

!iJ 500

mg/kg

*

.c ~ 60 E 40 20

o

Vehicle

Imipramine

Fig 1. Effect of the extracts of three Hypericum species on the total duration of immobility in the forced swimming test (FST) in mice. Results are means ± S.E.M., n=12, *p
The activities of the three Hypericum extracts on the tail suspension test are shown in Fig 2. The tricyclic antidepressant imipramine (20 mg! kg) reduced the immobility time significantly (43.2 ± 10.6 seconds from control 107.9 ± 10.4 seconds, p<0.05). H. erectum extract shortened the immobility time not significantly at 250 mg/kg (86.0 ± 16.4 s), and significantly at 500 mg/kg (73.0 ± 8.9 s). H. hubeiense extract shortened the immobility time significantly at 250 mg/kg (74.2 ± 9.0 s), but not

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244

significantly at 500 mg/kg (78.5 ± 12.5 s). H. seniavinii at 250 and 500 mg/ kg shortened the immobility time significantly (71.7 ± 11.2, 53.0 ± 9.3 s respectively). 140

D 2S0mg/kg

ISlSOOmg/kg

::§: 120 ~ 100 :;:::; 80

~ :c

60 o E 40 E 20

o

L -_ _~-L~~~_ _~~L-L-~~~~______-L~

Vehicle

HE

HH

HS

Imipramine

Fig 2. Effects ofthe extracts ofthree Hypericum species on the total duration of immobility in the tail suspension test (TST) in mice. Results are means ± S.E.M., n=12, *p<0.05, **p
5-HT - Induced Head Twitches in Mice In order to understand the mechanism of actions of antidepressant effects, the 5-HT - induced head twitches and potentiation of NA toxicity models were performed. The effects of the extracts of three Hypericum species on 5-HT induced head twitches are shown in Table 1, the extracts of H. erectum (250 and 500 mg/kg), H. hubeiense (250 mg/kg) and H. seniavinii (500 mg/ kg) produced significant potentiation of the head twitches, while imipramine did not produce significant effect at the dose of 20 mg/kg. In the control group there was no mice died, but in all the extract groups and imipramine group, the mice died at different death rates. The number of head twitches was counted from the survival mice. Table 1. Effects of the extracts of three Hypericum species on 5-HT induced head twitches (means ± S.E.M) Treatment Vehicle H. erectum H. seniavinii H. hubeiense

Imipramine

Dose (mglkg)

250 500 250 500 250 500 20

*p<0.05, **p
No. of head twitches 2.0 30.7 10.8 3.8 7.1 6.9 2.5 3.3

± ± ± ± ± ± ± ±

0.4 10.1** 0.7** 1.9 0.8* 0.7* 0.9 1.8

Rate of death 0/12 4/10 1110 3/10 2.110 1110 2.110 2.110

Antidepressant Activity of the Extracts of Hypericum Species

245

Potentiation ofNA Toxicity No death of mice was observed in control group at the sub-lethal dose (3 mglkg, sc.) ofNA. The results, as shown in Table 2, demonstrated that only H. hubeiense extract (500 mglkg) produced the potentiation of norepinephrine toxicity, similar to the control amitriptyline, whereas H. seniauini and H. erectum (250 and 500 mglkg) did not have the potentiation effect after acute administration. Table 2. Potentiation ofNA (3 mg/kg, sc.) toxicity in mice (means ± S.E.M, n=12) Dose (mg/kg)

Treatment Vehicle H. erectum

Rate of death

o o o o o o

250 500 250 500 250 500 20

H. seniavinii H. hubeiense Amitriptyline

3/12** 5/12**

*p<0.05, **p
Elevated Plus-Maze The results of the anxiolytic activity of the extracts were shown in Table 3. H. erectum, H. hubeiense extracts (250 and 500 mglkg) and the anxiolytic benzodiazepine drug, diazepam significantly increased residence time of animals in open arms and the number of entries to open arms. At the dosage Table 3. Effects of the extracts of three Hypericum species in the elevated plus-maze (EPM) test in mice (means ± S.E.M, n=12) Open arms Treatment

Dose (mg/kg)

Entries (N)

Vehicle H. erectum

H. seniavinii H. hubeiense Diazepam

250 500 250 500 250 500 3

5.2 8.7 11.6 7.0 6.8 10.2 9.3 9.6

± 0.7 ± 1.3* ± 3.1** ± 3.4 ± 1.8 ± 2.1** ± 1.3* ± 2.0*

*p<0.05, **p
Close arms

Time spent (s) 27.2 40.7 43.3 35.8 35.7 67.7 40.3 62.5

± 5.6 ± 12.3* ± 19.1* ± 6.9 ± 7.0 ± 16.6** ± 18.9* ± 11.0**

Entries (N)

11.3 12.8 11.4 9.7 14.8 10.1 10.2 12.8

± 2.1 ± 3.1 ± 3.7 ± 2.1 ± 3.1 ± 2.5 ± 4.1 ± 2.6

Time spent (s) 233.5 199.2 128.0 190.6 206.7 130.7 129.9 133.1

± 15.8 ± 19.5 ± 13.0** ± 20.2 ± 34.1 ± 10.6**

9.5** ± 8.0** ±

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of 500 mg/kg the two extracts decreased residence time in enclosed arms significantly. However, there was no significant change observed in the number of entries to the closed arm for the control and all extracts. The other species H. seniavinii extract (250 and 500 mg/kg) did not significantly affect the residence time and the number of entries in open arms and closed arms.

DISCUSSION The forced swimming test (FST) (Porsolt et al., 1977) and the tail suspension test (TST) (Steru et al., 1985) are the typical behavioral models commonly used in rodent for screening the antidepressant drugs. The immobility displayed in rodents, subjected to an unavoidable and inescapable stress, is known to reflect the behavioral despair, which may in turn reflect depressive disorders in humans (Goodwin et al., 1984; Alpermann et al., 1992). We studied the hydroalcoholic extracts of three Hypericum species endemic to China using these behavioral despair test models in mice. The results indicated that all the extracts (at the doses of 250 or 500 mg/kg, or both), and the conventional TCA antidepressants imipramine and amitriptyline, reduced the immobility time significantly. Serotonin and norepinephrine are known to play key roles in depression occurrences. Drugs that inhibit the reuptake of serotonin and norepinephrine are known to be effective in the treatment of major depression. H. hubeiense, H. seniavinii and H. erectum extracts could all significantly potentiate the 5-HT-induced head twitches compared with the control group. Additionally, all the extracts and imipramine induced partial mice death at different rates, acompanying with simultaneously drooling and frequent head twitches. These results indicate the possible actions of 5-HT reuptake inhibition (Shank et al., 1987) and potentiation of 5-HT toxicity in the serotoninergic system. It has been reported that some antidepressant drugs, such as, fiuoxetine, dexfenfluramine, may cause weight loss, along with their activities in increasing 5-HT availability in the synaptic clefts (McTavish et al., 1992; Goldstein et al., 1994). The weightlosing effect of H. seniavinii extract was observed in Limit Test, which also indicates the possibility of serotoninergic system involvement. The potentiation of norepinephrine toxicity in mice was examined to investigate the performance of the extracts in the noradrenergic system. In the experiment, 50% of the animals treated with amitriptyline died. However, only H. hubeiense extract could potentiate the norepinephrine toxicity at the dose of 500 mg/kg with 30% induced-mortality. H. erectum and H. seniavinii extracts showed no action in the noradrenergic system at the tested doses. No mortality occurred in the control group, neither. It suggested that the antidepressant activity of H. hubeiense extract maybe related to its possible effect in noradrenergic uptake inhibition.

Antidepressant Activity of the Extracts of Hypericum Species

247

Human depression is often accompanied by enhanced anxiety symptoms (Zimmernan et ai., 2002). There are reports showed that Hypericum also has anxiety activities besides its antidepressant effect. For example, Nicolas' experiments showed that Hypericum was active in preventing the anxiety effect of Mg-depletion animal model (Nicolas et al., 2004). Therefore, in current study, we also tested the potential anxiolytic effects of the hydroalcoholic extracts of these three Hypericum species in established anxiety test model of the elevated plus-maze. As demonstrated in Table 3, both H. hubeiense and H. erectum extracts exerted the anxiolytic activity in the elevated plus-maze similar to that caused by benzodiazepine drug diazepam. However, H. seniavinii did not show significant effect in anxiety improvement. The results indicate the possible involvement of H. hubeiense and H. erectum with benzodiazepine receptor. As the results indicate that the hydroalcoholic extracts of these three Hypericum species have antidepressant activities in mice models with different pattern of activities. H. hubeiense extract at the doses of 250 and 500 mg/kg significantly shortened the immobility time in FST, and significantly shortened the immobility time in TST at the dose of 500 mg! kg. It potentiated the head twitches induced by 5-HT, showed an increase of noradrenaline (NA) toxicity, and also showed anxiolytic activity in the elevated plus-maze test. These activities indicate that the possible mechanisms of action are inhibition of serotonine, and noradrenaline reuptake and binding of GABA receptor. It has the lowest toxicity among the three plants. The multiple mechanism of actions of H. hubeiense is resemble to H. perforatum, which has been shown to shorten the immobility behaviour, inhibit noradrenaline and serotonin re-uptake, inhibit monoamine oxidase, and interact with GABA receptor at a similar dosage (Butterweck et al., 2003).

H. erectum extract significantly shortened the immobility time in FST, and TST. It potentiated the head twitches induced by 5-HT, showed anxiolytic activity in the elevated plus-maze test, but did not increase noradrenaline (NA) toxicity. These activities indicate that the possible mechanisms of action are inhibition of serotonine re-uptake, and binding of benzodiazepine receptor, but not noradrenaline re-uptake. It has relatively high toxicity among the three plants. H. seniavinii extract significantly shortened the immobility time in FST, and TST. It potentiated the head twitches induced by 5-HT, but did not increase noradrenaline (NA) toxicity, or show anxiolytic activity in the elevated plus-maze test. These activities indicate the possible mechanism of action is inhibition of serotonine re-uptake, but not noradrenaline reuptake and binding of benzodiazepine receptor. The dose responses at 250 and 500 mg/kg were not observed for the TST activity of H. erectum extract, and potentiation of head twitches by H.

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hubeiense, while the activities ofthe two extracts in FST and elevated plusmaze test were dose-dependent. These phenomena may be due to the complexicity in chemical composition and pharmacological activities of the herbal extracts. The inverted V-shaped dose-dependent curve has been observed in shortening of immobility by hyperforin (Butterweck et al., 2003). As shown in Table 4, the Hypericum species contain various amounts of hypericin, pseudohypericin and individual flavonoids. The highest content of hypericins and flavonoids (5.76 mg/kg, 40.89 mg/kg respectively) are in H. erectum extract, and the lowest contents are in H. seniavinii extract. Therefore there is no direct correlation of the hypericins content with the antidepressant activity. Further studies are required to define their chemical profiles as well as the relationship between the main constituents and the antidepressant activities. Table 4. Yields ofthe hydroalcoholic extracts ofthree Hypericum species and the contents of the main constituents in the extracts Content in extract

Hypericum

Yield of

species

extract (%)

Hypericins HPLC

Flavonoids IN

H. hubeiense H. seniavinii

14.2 21.8

3.30 1.30

33.76 26.41

H. erectum

15.0

5.76

40.89

(mglg)

In summary the results suggest that these three Hypericum extracts possess antidepressant activity, which may be mainly mediated by serotonergic system. The H. hubeiense and H. erectum zalso possess anxiolytic activity. The results indicate that the three Chinese Hypericum species, in particularly H. hubeiense, possess pharmacological activities similar to H. perforatum and have the potential to be used as new source of antidepressant product.

ACKNOWLEDGEMENTS The authors extend their gratitude to Prof. Chang Hung-Ta and Dr. Wu Lihong for their contribution in identifying the plants, and to Prof. Chen Ruzhu and Prof. Zhu Xiaolan for their support in pharmacological experiments. The study was supported and funded by National Natural Science Foundation of China (2004 No.30470188); and partially supported by the Australia China Special Fund (2007).

REFERENCES Alpermann, H.G., Schacht, u., Usinger, P. and Hock, F.J. (1992). Pharmacological effects of Hoe 249: A new potential antidepressant. Drug Development Research, 25: 267-282.

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Butterweck, V., Christoffel, V., Nahrstedt, A., Petereit, F., Spengler, B. and Winterhoff, H. (2003). Step by step removal of hyperforin and hypericin: Activity profile of different Hypericum preparations in behavioral models. Life Sciences, 73: 627-639. Fu, P., Zhang, W.D., Liu, RH., Li, T.Z., Shen, Y.H., Li, H.L., Zhang, W. and Chen, H.S. (2006). Two new xanthones from Hypericum Japonicum. Natural Product Research, 20: 1237-1240. Goldstein, D.J., Rampey, A.H., Enas, G.G., Potvin, J.H., Fludzinski, L.A. and Levine, L.R (1994). Fluoxetine: A randomized clinical trial in the treatment of obesity. International Journal of Obesity and Related Metabolic Disorders, 8: 129-135. Goodwin, G.M., Green, A.R and Johnson, P. (1984). 5-HT2 receptor characteristics in frontal cortex and 5-HT2 receptor-mediated head twitch behavior following antidepressant treatment to mice. Brit£sh Journal of Pharmacology, 83: 235-242. Jiang, X., Williams, K.M., Liauw, W.S., Ammit, A.J., Roufogalis, B.D., Duke, C.C., Day, RO. and McLachlan, A.J. (2004). Effect ofSt John's wort and ginseng on the pharmacokinetics and pharmacodynamics of warfarin in healthy subjects. Brit£sh Journal of Clinical Pharmacology, 57: 592-599. Kasper, S. (2001). Hypericum perforatum-A review of clinical studies. Pharmacopsychiatry, 34(Suppl 1): 51-55. Linde, K., Ramirez,G., Mulrow, C.D., Pauls, A., Weidenhammer, W. and Melchart, D. (1996). St John's wort for depression-An overview and meta-analysis of randomized clinical trials. British Medical Journal, 313: 253-258. Lister, R. (1987). The use of a plus-maze to measure anxiety in the mouse. Psychopharmacology, 92: 180-185. McTavish, D. and Heel, RC. (1992). Dexfenfluramine: A review of its pharmacological properties and therapeutic potential in obesity. Drugs, 43: 713-733. Nicolas, S., Catrin, S., Alfred, H., Simone, B.S. and Harald, M. (2004). Magnesium- deficient diet alters depression- and anxiety-related behavior in mice-influence of desipramine and Hypericum perforatum extract. Neuropharmacology, 47: 1189-1197. Porsolt, RD., Le, P.M., Jalfre, M. and Chatterjee, S.S. (1977). Depression: A new animal model sensitive to antidepressant treatments. Nature, 266: 730-732. Sanchez-Mateo, C.C., Bonkanka, C.x., Prado, B. and Rabanal, RM. (2005). Antidepressant properties of some Hypericum canarlense L. and Hypericum glandulosum Ait. extracts in the forced swimming test in mice. Journal of Ethnopharmacology, 97: 541-547. Shank, R.P., Gardocki, J.F., Shcneider, C.R, Vaught, J.L., Settler, P.E., Marvanoff, B.E. and McComsey, D.F. (1987). Preclinical evaluation of McN-5707 as a potential antidepressant. Journal of Pharmacology and Experimental Therapeutics, 242: 74-84. Steru, L.R, Chermat, B. and Thierry, P., Simon. (1985). The tail Suspension Test: A new method for screening antidepressant activity in mice. Psychopharmacology, 85: 367-370. Willner, P. (1984). The validity of animal models of depression. Psychopharmacology, 83: 1-16. Wu, L.H., Hu, H.Y., Huang, S.L. and Yang, D.P. (2002). Review of historical literature records of Forsythia suspensa Vahl and Hypericum perforatum L. China Journal of Chinese Materia Medica, 27: 612-616. Wu, L.H., Yang, D.P. and Wang, F.S. (2004). New taxa of Hypericum (Hypericaceae) in China. Acta Phytotax Sin. 42: 3-8. Zheng, Q.M., Qin, L.P. and Zheng, H.C. (2003), Quantitative analysis of Chemical compositions in 11 Chinese Hypericum plants. Jounal of the Second Military Medicine University, 24: 457-459. Zimmernan, M., Chelminski, I. and McDermut, W. (2002). Major depressive disorder and axis I diagnostic comorbidity. Journal of Climcal Psychiatry, 63: 187-193.

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12 Cytolytic and Toxic Effects of Ostreolysin, a Protein from the Oyster Mushroom (Pleurotus ostreatus) KRISTINA SEPCIC 1 AND ROBERT FRANGEZ 2,*

ABSTRACT

Ostreolysin is a 15 kDa cytolytic protein found in considerable amounts in oyster mushrooms (Pleurotus ostreatus). Ingestion of large quantities of these edible mushrooms can result in adverse reactions, and at least some of these effects can be attributed to ostreolysin. This protein is able to permeabilize erythrocytes and other cells at sub-micromolar concentrations, acting by a colloid-osmotic mechanism and inducing the formation of a membrane pore with a hydrodynamic radius of 2 nm. Its binding to and subsequent permeabilization of the membrane depend on interaction with cholesterolenriched raft-like membrane domains. When injected intravenously into rodents, ostreolysin induces cardiorespiratory arrest and is lethal for mice with an LD50 of 1170 pg/kg. In rats, it induces a transient increase in arterial blood pressure, followed by a progressive drop of blood pressure, associated with noticeable bradicardia and myocardial ischemia. Continued drop of blood pressure is accompanied with ventricular extrasystoles, related to marked hyperkalemia. Moreover, sub-micromolar concentrations of ostreolysin induce a concentration-dependent increase in aortic ring tension. Key words : Cardiorespiratory effect, hemolysis, hyperkalemia, lipid rafts, ostreolysin, Pleurotus ostreatus, toxicity, pore forming protein 1. Department of Biology, Biotechnical Faculty, University of Ljubljana, Vecna pot 111, 1000 Ljubljana, Slovenia. 2. Institute of Physiology, Pharmacology and Toxicology, Veterinary Faculty, University of Ljubljana, Slovenia. * Corresponding author: E-mail: [email protected]

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INTRODUCTION Ostreolysin (Oly) is an acidic 15 kDa protein that is synthesized specifically in the primordia and young fruiting bodies of the oyster mushroom (Pleurotus ostreatus) (Berne et al., 2002). The protein belongs to a larger group of highly homologous proteins called aegerolysins. Aegerolysin-like proteins have been isolated from several mushrooms, moulds, plants and bacteria (Berne et al., 2005). They all have low isoelectric points, similar molecular weights, and are heat-labile. Furthermore, they are stable over a wide range of pH, mainly from 4 to 10 (Sakaguchi et al., 1975; Berne et al., 2005). Several aegerolysins are expressed during the formation of mushroom fruit bodies (Fernandez-Espinar & Labarere, 1997; Berne et al., 2002; Vidic et al., 2005) and during bacterial sporulation (Barloy et al., 1998), which suggests their involvement in the developmental cycle of the organism that produces them. Oly can induce the formation of primordia and development of young fruit bodies of P. ostreatus, even when applied exogenously to the mushroom mycelium (Berne et al., 2007). Binding to natural and artificial lipid membranes followed by permeabilization is another common feature of many aegerolysins. Asphemolysin from the mould Aspergillus fumigatus, as well as mushroomderived aegerolysins (aegerolysin from Agrocybe aegerita, Oly and pleurotolysin A (Ply A) from Pleurotus ostreatus, and eryngeolysin from Pleurotus eryngii) have been reported to lyse erythrocytes and various cell lines (Ebina et al., 1985; Berne et al., 2002; SepCic et al., 2003; Tomita et al., 2004; Ngai & Ng, 2006). The hemolytic and cytolytic effects of aegerolysin-like proteins appear to be associated with their toxicity. One of the most studied aegerolysins, Asp-hemolysin, can be considered as a virulence factor of A. fumigatus during infection of animals by this opportunistic pathogenic mould (Ebina et al., 1982; Malicev et al., 2007). The edible oyster mushroom, although being a massively cultivated species with important medicinal, biotechnological, and environmental applications (Kiies & Liu, 2000; Cohen et al., 2002) has been reported to induce sporadic local intoxications (AlDeen et al., 1987). These toxic effects appear to be connected with Oly, which causes cardiorespiratory arrest after intravenous administration to mice, with a half-lethal dose of 1170 pg/kg CZuzek et al., 2006). The lytic process caused by many pore-forming proteins starts by their recognition of a distinctive membrane component such as a membrane lipid. Aegerolysins show considerable variability in this respect. Asphemolysin, and recombinant PA0122 from Pseudomonas aeruginosa have been suggested to bind specifically to lysophosphatidy1choline (Kudo et al. , 2002; Rao et al., 2008), PlyA to sphingomyelin (SM) (Tomita et al., 2004) and Oly to sense specifically membrane domains enriched together in cholesterol

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(Chol) and SM (SepCic et al., 2004). Lateral cell membrane domains, known as lipid rafts, are highly enriched in these two lipids (Pike, 2004). These transient membrane entities, existing in a liquid-ordered (l) state (McConnell & Vrljic, 2003), are more resistant to solubilization by detergents than lipids in liquid-disordered (ld) domains (Lichtenberg et al., 2005) and are therefore often termed detergent-resistant membranes (DRMs) (Simons & Ikonen, 1997). Lipid rafts are involved in several important biological functions, including exocytosis and endocytosis, signal transduction, pathogen entry, and attachment ofligands (Simons and Ikonen, 1997; London, 2002; Edidin, 2003), and stabilize on binding ligand molecules (Kenworthy et al., 2000; Subczynski & Kusumi, 2003; Hancock, 2006). Due to their important functions in the living cell and in cell-cell interactions, there is an increasing need for new techniques to study lipid rafts. Oly, which recognizes specifically the combination of the two main lipid raft components, SM and Chol, is in this regard a good candidate for a new marker of Chol-enriched raft-like membrane microdomains.

MEMBRANE ACTIVITY OF OSTREOLYSIN The ability to bind to natural and artificial lipid membranes and form pores is a common feature of aegerolysin-like proteins. There have been several attempts to express recombinant aegerolysins in bacterial hosts, however most were devoid oflytic potential (Juarez-Perez & Delecluse, 2001; Kumagai et al., 2002; Rao et al., 2008; Rebolj & SepCic, unpublished data). The sole exception is the recombinant form of PlyA, which acts as an active bicomponent cytolysin in concert with the recombinant 59 kDa pleurotolysin B (PlyB) (Sakurai et al., 2004).

Hemolytic and Cytolytic Activities Oly lyses erythrocytes from a variety of species at sub-micromolar concentrations (SepCic et al., 2003). The time course of hemolysis is typical of pore-forming proteins that bind to the membrane in their monomeric form, oligomerize, insert and translocate their polypeptide segments across the lipid bilayers, resulting in the formation of a transmembrane pore consisting of several protein monomers (Gouaux, 1997). The hydrodynamic radius of an Oly-induced transmembrane pore, determined by the use of different osmotic protectants, is 2 nm (SepCic et al., 2003). Some aegerolysins have been shown to form rings and patches at the surface of erythrocyte membranes, detected by scanning electron microscopy (Ebina et al., 1985; Tomita et al., 2004). In the case of pleurotolysin, SDS-stable, ring-shaped 700 kDa complexes were observed when Ply A and Ply B were mixed in a 3:1 molar ratio (Tomita et al., 2004). Incubation of Oly with lipid vesicles formed from total sheep erythrocyte lipids, followed by ultracentrifugation, showed that Oly is located exclusively in the sediment, also pointing to its

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tight binding with lipid membranes. Furthermore, SDS-PAGE analysis of the diluted sediment showed the formation of membrane-bound Oly aggregates of around 34, 64, and 100 kDa, corresponding to dimers, tetramers, and hexamers (SepCic et al., 2003). The pH-optima for Oly binding to the erythrocyte membrane, and for the consequent permeabilization, differ. Optimal binding is usually achieved between pH 6 and 7, while maximal hemolysis is observed between pH 7 and 8 (Berne et al., 2005). Oly however retains its membrane activity over a wide range of pH, from 3.5 to 10.5 (Berne et al., 2005). At physiological pH, the binding of Oly to a membrane correlates with an increase in its intrinsic tryptophan fluorescence and a-helical content (SepCic et al., 2003). Fluorescence titration experiments revealed that Oly changes its conformation more extensively in the lipid-bound than in the free form, suggesting that binding to the bilayers evokes further conformational transitions necessary for membrane insertion and pore formation (Berne et al., 2005). Further, the involvement of intact Oly cysteine residues in the hemolytic process has been proved to be important (Berne et al., 2002, 2005). Lysis can be prevented by micromolar concentrations of some divalent ions, like Hg2+ and Fe 2+ (Berne et al., 2002). Finally, Oly-induced hemolytic effects are abolished by the addition of micromolar concentrations of lysophospholipids (especially lysophosphatidylinositol and sphingosine-I-phosphate), fatty acids (SepCic et al., 2003), and lipid vesicles composed of an equimolar SM:Chol mixture (SepCic et al., 2004). The differing sensitivity of erythrocytes from different species to aegerolysins is another interesting feature (Sakaguchi et al., 1975; Bernheimer & Avigad, 1979; Tomita et al., 2004; Ngai & Ng, 2006). Oly showed no difference in its ability to lyse sheep, bovine and human erythrocytes (SepCic et al., 2003) but was much less potent on those from rodents (Zuiek et al., 2006), dog, and amphibians (Rebolj & SepCic, unpublished data) (Fig 1). In addition to erythrocytes, Oly and several other aegerolysins have been found to be cytotoxic for cell lines at sub-micromolar concentrations (SepCic et al., 2003; SepCic et al., 2004; Malicev et al., 2007; Rebolj et al., 2007) (Table 1). Direct microscopic observation of some tumour cells exposed to Oly showed typical signs of colloid-osmotic lysis, like swelling, blebbing and degranulation of cells (SepCic et al., 2003). Similar events were observed during real-time monitoring of Oly-induced disruption of fluorescently stained chondrocytes and osteoblasts, using confocal microscopy (Malicev et al., 2007). Lytic effects of Oly on different cells can induce hyperkalaemia and are therefore, at least partially, responsible for the observed toxicity of the protein after intravenous administration (Zuiek et al., 2006).

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Fig 1. Hemolytic activity of ostreolysin on erythrocytes. Hemolysis was assayed by a turbidimetric method (Sepcic et ai., 2003). The reciprocal half-time of hemolysis, (lIt50 ), is plotted against the concentration of ostreolysin, Oly (0.0055 to 3 }lIIl). t50 is the time in which half of the erythrocytes were lysed. Each result is the mean±standard error of three independent experiments Table 1. Cytotoxic properties ofOly on cell lines. ED50 is the concentration of ostreolysin producing 50% cytotoxicity Cell line

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Reference(s)

HT 1080 (fibrosarcoma, human) MCF 7 (mammalian tumour, human) Chinese hamster ovary cells Human chondrocytes V-79-379A (lung fibroblasts of Chinese hamster) HUVEC (human umbilical vein endothelial cells)

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Mode of Ostreolysin Binding to Cell Membranes In order to elucidate the structural characteristics of Oly-susceptible membranes, we performed an extensive preliminary study using artificial lipid membranes. Lipid monolayers or bilayers, the latter in the form of vesicles of different sizes, with a range of lipid compositions, were tested for their susceptibility to Oly. Binding was assessed either indirectly in a hemolysis-inhibition assay by incubating Oly with lipid vesicles and subsequent addition of erythrocytes (SepCic et ai., 2003), or directly, using surface plasmon resonance on supported lipid monolayers (SepCic et ai., 2004; Rebolj et ai., 2006; Chowdhury et ai., 2008). Membranepermeabilizing activity was assessed mainly by measuring the leakage of the fluorescent dye calcein from lipid vesicles following the addition of

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Oly (SepCic et al., 2003; Sepcic et al., 2004; Rebolj et al., 2006). We found that lipid vesicles composed of equimolar mixtures of egg phosphatidiylcholine with various other lipids were completely resistant to Oly binding and permeabilization (Sepcic et al., 2003). The only vesicles showing susceptibility to Oly membrane activity were those composed of SM and Chol in a 1: 1 molar ratio (Sepcic et al., 2004). Due to the facts that (i) Oly cannot bind to pure SM or Chol, and (ii) the two lipids are known to be highly enriched in lipid rafts, Oly specific interaction with SM:Chol liposomes suggested its interaction with Chol-enriched raft-like membrane micro domains in living cells. This finding was confirmed by a series of experiments. First, Oly was found in isolated DRMs of both SM:Chol (1:1, mol:mol) vesicles and Chinese Hamster Ovary (CHO) cells (Sepcic et al., 2004). Secondly, Oly bound to and permeabilized SM:Chol membranes in various molar proportions of the two lipids, but only above 30 mol% Chol (Sepcic et al., 2004, Rebolj et al., 2006), the concentration at which this sterol induces the formation of a liquid-ordered phase (de Almeida et al., 2003). Further, binding of sub-lytic concentrations of Oly to various mammalian cells and cell lines (CHO cells, mouse somatotrophs, human chondrocytes and osteoblasts), followed by immunolocalization using fluorescence-labeled antibodies, showed selective binding and clustering of the protein on cell membranes, pointing to its association with distinctive membrane microdomains rich in Chol (Malicev et al., 2007, Chowdhury et al., 2008). Lipid rafts are sensitive to treatment with methyl-~-cyclodextrin (M~CD), which acts as a Chol scavenger and promotes disruption of the structural and functional integrity oflow density membrane domains (Pike & Miller, 1998). In CHO cells pre-treated with 20 mM M~CD (and depleted by 43% of their Chol content), Oly membrane binding was dramatically decreased to only 2%. However, treatment with 10 mM M~CD, which resulted in depletion of membrane Chol by 35%, caused no significant reduction in the binding of Oly (Chowdhury et al., 2008) (Fig 2). In accordance with the results obtained with SM:Chol vesicles in various molar ratios (Sepcic et al., 2004), these data suggest that, to effectively bind and permeabilize lipid membranes, Oly requires a specific threshold membrane Chol concentration. The same trend was observed in experiments with CHO cell mutants having impaired Chol synthesis and consequently a lower fraction of raft-like membrane domains. Binding of Oly to these cells clearly correlated with the amount of their membrane Chol (Sepcic et al., 2004). However, not all our results suggest Oly as a classical lipid raftbinding protein. Our experiments using lipid mono- and bi-Iayers composed of SM and various sterols and Chol derivatives revealed that Oly membrane activity does not completely correlate with the ability of a particular steroid to induce the formation of lo domains (Rebolj et al.,

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2006). Further, Oly cannot exert its membrane activity on lipid membranes composed of the equimolar mixture of SM, Chol and unsaturated glycerophospholipids that are generally used to mimic the membranes of living cells (SepCic et al., 2004). Finally, immunostaining studies using CHO cells showed that Oly was not co-localized with typical raft-binding proteins like caveolin and cholera toxin B-subunit, suggesting that it binds to distinct Chol-enriched membrane domains (Chowdhury et al., 2008). Altogether, these findings reinforce the emerging idea of the microheterogeneity of the lipid rafts (Roper et al., 2000; de Almeida et al., 2005; Hancock, 2006), comprising at least 2 Chol-enriched domain types - cave olin-containing (caveolae), and caveolin-Iacking rafts (Pike, 2004). It appears that Oly, rather than recognizing the pure lipid component (e.g. Chol or SM), or a specific physical state of the membrane (e.g. a liquid-ordered state), recognizes and binds to specific patterns that are formed by Chol molecules at the surface of biological membranes. This conclusion is reinforced by the fact that the addition of Oly does not affect the electron paramagnetic resonance spectra of equimolar SM:Chol vesicles, which would point to the recruitment of the lipids after the ligand binding (Sepcic et al., 2004).

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WHOLE ANIMAL TOXICITY STUDIES OF OSTREOLYSIN AND IN VITRO STUDIES ON TIlE CELL AND ORGAN LEVEL

In vivo Effects of Ostreolysin Despite its putative physiological role during the formation of mushroom fruit bodies (Berne et al., 2002; Vidic et al., 2005; Berne et al., 2007), experiments on erythrocytes and artificial membranes revealed that Oly is also a pore-forming agent (Sepcic et al., 2003). The hemolytic and cytolytic activity of many bioactive proteins from different sources appears to be associated with their toxicity. Many questions arise as to whether these properties are also responsible for Oly toxicity, as demonstrated for another aegerolysin-like protein, Asp-hemolysin (Ebina et al., 1982; Malicev et al., 2007). To investigate this, the toxic and lethal effects of Oly were further studied on experimental rodents in vivo and in vitro. The lethal and toxic effects, expressed as LD50 dose, have been determined in mice and rats, respectively. To determine LD50 , Oly was dissolved in physiological solution and injected as a bolus through the right tail vein of mice. Subsequent clinical signs comprised cyanosis, cessation of movement and hair bristle, and lethal outcome within 20 min for all mice at a dose of 1400 llg/kg or higher CZuiek et al., 2006). The i.v. LD50 in mice was 1170 J.Ig!kg (Zuiek et al., 2006). In order to study its effects on the cardiovascular system and systemic response, in vivo experiments with Oly were performed on adult male Wistar rats. Oly was dissolved in physiological solution and applied as a bolus through the cannulated right jugular vein. Respiratory activity, electrocardiograms (ECG) and blood pressure were monitored. Since Oly is lytic to erythrocytes from various animal species and cell lines (Fig 1), some blood electrolytes were also monitored to find out whether the heart rhythm abnormalities caused by Oly are related to the altered ion concentration (in particular to potassium concentration related to its hemolytic action). In the sub-micromolar range, the toxin lysed rat erythrocytes in vitro in a dose-dependent manner. When injected into rats under general anesthesia, one mice LD50 of Oly causes death in all experimental animals, suggesting that rats are more sensitive than mice. Hemolysis, with a resulting rise in plasma potassium concentration, is probably an important factor in its toxic and lethal effects, as already shown for some other pore forming toxins (Suput et al., 2001; Andrade et al., 2004). In a dose of one mice LD50 , Oly produced a transient pressor response in rats, followed by a prolonged hypotensive response and a progressive, irreversible fall of blood pressure to its mid-circulatory value. In addition, transient apnea is observed after injection of Oly, followed by transient respiratory activity and respiratory arrest (Zuzek, Sepcic & Frangez, unpublished results) (Fig 3). The transient pressor response induced by 1 LD50 of Oly lasted for approximately 1-3 min. The basal mean arterial

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blood pressure (MAP) of anaesthetized rats increased from 87 ± 7.6 mm Hg (n=6) before the injection ofOly to 116 ± 9.7 mm Hg (n=6) afterwards. The pressor response was followed by a progressive and irreversible fall in arterial blood pressure (aBP) to the mid-circulatory blood pressure (6-8 mmHg) in all experiments (Fig 3). Oly produced a respiratory arrest in 10-20 s after the toxin injection, followed by a transient respiratory activity after 1-2 min. Two min after injection into anaesthetized rats, Oly significantly reduced the heart rate from 266 ± 14.6 to 121 ± 21.9 (p$;0.05, n=6). At the same time, changes in ECG appeared as sinus arrhythmia, with peaking and increased amplitude of the T-wave, and increased prolonged P-R interval. Elevation ofthe S-T segment, ventricular extrasystoles and widened QRS complexes were also observed regularly, suggesting myocardial ischemia (Fig 3) (Zuzek et al., 2006). Oly binds specifically to raft-like Chol-rich membrane domains (SepCic et al., 2004; Rebolj et al., 2006), followed by the formation of 4 nm transmembrane pores (SepCic et al., 2003). Severe hyperkalemia (10.28 ± 1.25 mmolll, p$;0.05, n=6), caused by the lytic action of Oly on erythrocytes and other cells, may be responsible for bradycardia and irregular heart rate and rhythm in rats (Zuiek et al., 2006). To evaluate the role of cholinergic stimulation of the heart, potentially triggered by the action of Oly on arterial blood pressure and on the ECG, its effects were studied in atropinized rats. Although the effects of Oly on heart rate were less pronounced than in the control group of rats, the muscarinic blocker, atropine, did not significantly abolish or alter the cardiorespiratory effects described for the non-atropinized group of animals injected with Oly. It was therefore concluded that parasympathetic stimulation of the heart plays only a minor role in Oly cardiotoxic action (Zuzek et al., 2006). It was also found that Oly produces the same changes in ECG, blood pressure, and biochemical blood parameters in artificially ventilated rats (n=4), which excludes hypoxia as the primary cause of cardiotoxic effects initiated by respiratory arrest (Zuiek et al., 2006). Although hyperkalemia, suggesting in vivo lysis of erythrocytes and other cells, may be significant for its lethal action, the exact mechanism of the lethal effect of Oly remains unclear. Results from the in vivo experiments (arrhythmias, drop of blood pressure, elevation of S-T segment) suggest myocardial hypoxia as the primary effect. Hence, in order to identify the cellular and molecular mechanisms of the toxic action responsible for in vivo effects of Oly, its effects on vessel ring tension and endothelial cell viability have recently been studied (Rebolj et al., 2007).

Effect of Oly on Isolated Coronary Rings To identify its target organs and cells, in vitro experiments with Oly have been performed on thoracic aortic ring preparations, human umbilical vein

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Compo Bio. Nat. Prod., Vol. 2 -Efficacy, Safety & Clinical Evaluation I Oly A~3

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endothelial cells (HUVEC), and lung fibroblasts of Chinese hamster (V-79379A). Oly induced concentration-dependent rat aorta ring contractions at final concentrations of 1 to 50 JJglmL. After the aorta rings were exposed to various concentrations of Oly, endothelium-mediated relaxation of rings pre-contracted with norepinephrine was diminished. This effect may be due to injury of the endothelial cells or to the breakdown of the mechanisms responsible for the acetylcholine (ACh) mediated endothelium relaxation response (Rebolj et al., 2007). This result is in agreement with those related to the lytic effects of O1y on erythrocytes (Sepcic et al., 2003) and umbilical vein endothelial cells (Rebolj et al., 2007). The effective cytotoxic concentration (EC 50 ) of O1y evaluated after 48 h was 1.3 JJglmL for V-79, and 2.2 JJglmL for HUVEC cells in the growth medium (Rebolj et ai., 2007).

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The direct damage of endothelial cells probably plays an important role in the cardiotoxicity of Oly CZuzek et al., 2006). This view is additionally supported by the results obtained from the experiments with aortic rings having preserved endothelium, where a functionally defective ACh-mediated relaxation response was clearly demonstrated CRebolj et al., 2007). The final concentrations ofOly (15-30 llg/mL) in the bathing medium, which produced a significant increase in aorta ring tension in vitro, were similar to the maximal calculated concentrations of Oly in the blood during the in vivo experiments. It is suggestive that especially the coronary vessels contraction may be important as the underlying cause of death in experimental rodents (Rebolj et al., 2007). The same effects on coronary vessels could result in a vasospasm leading to ischemia and hypoxic injury of cardiomyocytes. This view is also supported by the ECG data from in vivo experiments CZuzek et al., 2006). Direct harmful effect of Oly on cardiomyocytes and cells of the conductive system of the heart also cannot be excluded.

CONCLUSIONS AND PERSPECTIVES Since Oly may be a causative agent responsible for some documented adverse responses associated with eating oyster mushrooms, it is important to reveal the exact molecular mechanisms responsible for its cardiorespiratory and lethal action observed in rodents. Moreover, although its physiological role in the mushroom is still uncertain, Oly displays a variety of effects that could have valuable applications in various fields of human activity. First, its ability to trigger the fruiting of oyster mushrooms after its application to the mushroom mycelium (Berne et al., 2007) could be used in the field of biotechnology. Secondly, because of its specific interaction with Chol-rich raft-like membrane domains (Septic et al., 2004; Malicev et al., 2007; Chowdhury et al., 2008), Oly could find its place in the field of cell biology as a lipid raft marker. Fluorescence- or spin-labeled raft-binding mutants of cytolytic proteins, devoid of their lytic activity, are good candidates in this regard. One of these (the GM1 ganglioside-binding cholera-toxin B subunit (Bacia et al., 2004) forms part of a commercially available raft-labeling kit. Finally, the cytolytic and hemolytic properties of Oly are comparable to those exerted by Asp-hemolysin, the putative virulence factor of the pathogenic mould Aspergillus fumigatus (Malicev et al., 2007). Studies of the molecular mechanisms of Oly action could therefore shed some light on the action of Asp-hemolysin during the infection, and help in the development of vaccines or in the use of antibodies against infection by A. fumigatus spores, which is very frequent in hospitals associated with surgery. ACKNO~DGEMENTS

The authors gratefully acknowledge Prof. Roger Pain for critical reading of the manuscript, and the Slovenian Research Agency for the financial support.

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Kenworthy, A.K., Petranova, N. and Edidin, M. (2000). High-resolution FRET microscopy of cholera toxin B-subunit and GPI-anchored proteins in cell plasma membranes. Molecular Biology of the Cell, 11: 1645-1655. Kiies, D. and Liu, Y. (2000). Fruiting body production in basidiomycetes. Applied Microbwlogy and Biotechnology, 54: 141-152. Kudo, y., Ootani, T., Kumagai, T., Fukuchi, Y. and Ebina, K (2002). A novel oxidized lowdensity lipoprotein binding protein, Asp-hemolysin, recognizes lysophosphatidylcholine. Biological & Pharmaceut£cal Bulletin, 25: 787-790. Kumagai, T., Kudo, Y., Fukuchi, Y., Ebina, K. and Yokota, K. (2002). Expression of a synthetic gene encoding the Asp-hemolysin from Aspergillus fum£gatus in Eschenchia coli. Biological & Pharmaceutical Bulletin, 25: 115-7. Lichtenberg, D., Goni, F.M. and Heerklotz, H. (2005). Detergent-resistant membranes should not be identified with membrane rafts. Trends in Biochem£cal Sciences, 30: 430-436. London, E. (2002). Insights into lipid raft structure and formation from experiments in model membranes. Current Opinion in Structural Biology, 12: 480-486. Malicev, E., Chowdhury, H., Macek, P. and SepCic, K. (2007). Effect of ostreolysin, an Asp-hemolysin isoform, on human chondrocytes and osteoblasts, and possible role of Asp-hemolysin in pathogenesis. Medical Mycology, 45: 123-130. McConnell, H.M. and Vrljic, M. (2003). Liquid-liquid immiscibility in membranes. Annual Review of Biophysics & Biomolecular Structure, 32: 469-492. Ngai, P.H.K. and Ng, T.B. (2006). A hemolysin from the mushroom Pleurotus eryngii. Apphed M£crobiology and Biotechnology, 72: 1185-1191. Pike, L.J. (2004). Lipid rafts: heterogeneity on the high seas. Bwchem£cal Journal, 378: 281-292. Pike, L.J. and Miller, J.M. (1998). Cholesterol depletion delocalizes phosphatidylinositol bisphosphate and inhibits hormone-stimulated phosphatidylinositol turnover. Journal of Biolog£cal Chemistry, 273: 22298-22304. Rao, J., DiGiandomenico, A., Dnger, J., Bao, Y., Polanowska-Grabowska, R.K. and Goldberg, J.B. (2008). A novel oxidized low-density lipoprotein-binding protein from Pseudomonas aeruginosa. Microbiology, 154: 654-665. Rebolj, K., Poklar Dlrih, N., Macek, P. and Sepcic, K. (2006). Steroid structural requirements for interaction of ostreolysin, a lipid-raft binding cytolysin, with lipid monolayers and bilayers. Biochimica et Bwphysica Acta, 1758: 1662-1670. Rebolj, K., Batista, D., SepCic, K., Cestnik, V., Macek, P. and Frangez, R. (2007). Ostreolysin affects rat aorta ring tension and endothelial cell viability in vitro. Toxicon, 49: 1211-1213. Roper, K., Corbeil, D. and Huttner, W.B. (2000). Retention of prominin in microvilli reveals distinct cholesterol-based lipid micro-domains in the apical plasma membrane. Nature Cell Biology, 2: 582-592. Sakaguchi, 0., Shimada, H. and Yokota, K. (1975). Purification and characteristics of hemolytic toxin from Aspergillus fumigatus. Japanese Journal of Medical Sciences and Biology, 28: 328-331. Sakurai, N., Kaneko, J., Kamio, Y. and Tomita, T. (2004). Cloning, expression, and poreforming properties of mature and precursor forms of pleurotolysin, a sphingomyelinspecific two-component cytolysin from the edible mushroom Pleurotus ostreatus. Bwchimica et Biophysica Acta, 1679: 65-73. SepCic, K., Berne, S., Potrich, C., Turk, T., Macek, P. and Menestrina, G. (2003). Interaction of ostreolysin, a cytolytic protein from the edible mushroom Pleurotus ostreatus, with lipid membranes and modulation by lysophospholipids. European Journal of Bwchemistry, 270: 1199-1210. SepCic, K., Berne, S., Rebolj, K., Batista, D., Plemenitas, A., Sentjurc, M. and Macek, P. (2004). Ostreolysin, a pore-forming protein from the oyster mushroom, interacts specifically with membrane cholesterol-rich lipid domains. FEBS Letters, 575: 81-85. Simons, K and Ikonen, E. (1997). Functional rafts in cell membranes. Nature, 387: 569-572. Subczynski, W.K. and Kusumi, A. (2003). Dynamics of raft molecules in the cell and artificial membranes: approaches by pulse EPR spin labeling and single molecule optical microscopy. Bwchimica et Biophystca Acta, 1610: 231-243.

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Suput, D., Frangez, R. and Bunc, M. (2001). Cardiovascular effects of equinatoxin III from the sea anemone Actmia equina (L.). Toxicon, 39: 1421-1427. Tomita, T., Noguchi, K., Mimuro, H., Ukaji, F., Ito, K., Sugawara-Tomita, N. and Hashimoto, Y. (2004). Pleurotolysin, a novel sphingomyelin-specific two-component cytolysin from the edible mushroom Pleurotus ostreatus, assembles into a transmembrane pore complex. Journal of Biological Chemistry, 279: 26975-26982. Vidic, I., Berne, S., Drobne, D., Macek, P., Frangez, R., Turk, T., Strus, J. and SepCic, K. (2005). Temporal and spatial expression of ostreolysin during development of the oyster mushroom (Pleurotus ostreatus). Mycological Research, 109: 377-382. Zuzek, M.C., Macek, P., SepCic, K., Cestnik, V. and Frangez, R. (2006). Toxic and lethal effects of ostreolysin, a cytolytic protein from edible oyster mushroom (Pleurotus ostreatus), in rodents. Toxicon, 48: 264-271.

13 Antioxidant, Antisecretory and Gastroprotective Activities from Leiothrix flavescens THIAGO DE MELLO MORAES 1, MARCELO APARECIDO SILVA2, CLENILSON MARTINS RODRIGUES 2, LOURDES CAMPANER DOS SANTOS 2, MIRIAM SANNOMIYA2 , LUCIA REGINA MAcHADO DA ROCHA 1, ALBA REGINA MONTEIRO SOUZA BRIT03 , TAIS M. BAUAB 4 , WAGNER VILEGAS 2 AND CLl!;LIAAKIKO HIRVMA-LIMAl,*

ABSTRACT Leiothrix flavescens, popularly know as "sempre viva" belongs to Eriocaulaceae family with important biological activity. Extracts of this specie contain mainly flavones and xanthones. The methanolic extract (MeOH) obtained the scapes of L. flavescens was evaluated for its ability to protect the gastric mucosa against gastric mucosa injuries agents in rodents such as HClIEtOH, non-steroidal anti-inflammatory drug or stress. We also evaluated its activity against Helicobacter pylori and its antioxidant property. All pretreatment with MeOH extract from L. flavescens show significant gastroprotective action. No acute toxicity was observed in animals treated with MeOH extract at doses of 5 g/kg (p.o.). The MeOH extract from L. flavescens also increased intestinal motility and changed gastric juice parameters significantly by decreasing gastric acid secretion and promoting reduced acid output. The MeOH extract also shown effective action against H. pylori by inhibiting their growth and presented an interesting antioxidant action in vitro. The 1. Physiology Department, Biosciences Institute, Sao Paulo State University-UNESP, c.p. 510; Zip Code: 18618-000, UNESP, Botucatu, SP, Brazil. 2. Organic Chemistry Department, Chemistry Institute, Sao Paulo State UniversityUNESP, c.p. 355; Zip Code: 14800-900, UNESP, Araraquara, SP, Brazil. 3. Physiology and Biophysics Department, Biology Institute-UNICAMP, c.p. 6109; Zip Code: 13083-970, UNICAMP, Campinas, SP, Brazil. 4. Biological Sciences Department, Sao Paulo State University-UNESP, c.p.355; Zip Code: 14800-900, UNESP, Araraquara, SP, Brazil. * Corresponding author: E-mail: [email protected].

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present data show the effectiveness of MeOH extract from L. fiavescens as antacid/antiulcer and laxative. Key words: Flavonoids, antiulcer, Leiothrix fiavescens, xanthones, antiHelicobacter pylori, antioxidant action

INTRODUCTION Leiothrix Ruhland is an exclusively South American genus of about 37 species mainly restricted to Brazil, with only two species occurring elsewhere: Leiothrix fiavescens (Bong.) Ruhland that occurs in Brazil, Venezuela, Guyana and Peru, and Leiothrix celiae Moldenke which is found only in Venezuela (Giulietti et al., 1998). The other species are mostly endemic to small areas in Minas Gerais and in Bahia, states of central Brazil (Giulietti et al., 1994). This genus and other species of the Eriocaulaceae family such as Syngonanthus and Paepalanthus are known as "sempre vivas". Chemical studies on species of Leiothrix are also scarce. Salatino et al. (1990) studied the contents of soluble phenolic compounds of the capitula of 21 species of Leiothrix. The presence of nepetin-7 -O-P-Dglucopyranoside, nepetin-7 -O-P-D-arabinopyranoside and luteolin 0- and C-glucopyranoside were determined in Leiothrix species (Dokkedal & Salatino, 1992). Another study determined the presence of xanthones and flavonoids with antioxidant activity in capitula of Leiothrix species (Santos et al., 2001). Although there has been no pharmacological study about this species, the Eriocaulaceae family has presented significant effects on gastrointestinal tract. The antiulcerogenic activity of two species of Syngonanthus (Coelho et al., 2006; Batista et al., 2004) has also been determined. Di Stasi et al. (2004) reported the intestinal anti-inflammatory activity of paepalantin, an isocoumarin isolated from Paepalanthus bromelioides (Eriocaulaceae).Therefore, the present work was carried out in order to investigate the gastric effect of the methanolic extract (MeOH) from scapes of L. fiavescens against acute experimental models of gastric ulcer in rodents as well as to perform its phytochemical investigation.

MATERIALS AND METHODS Plant Material Scapes of Leiothrix fiavescens (Bong.) Ruhland were collected in May 1997, at Serra do Cipo, Minas Gerais state, Brazil and authenticated by Professor Dr. Paulo Takeo Sano from Instituto de Biociencias (IB) da Universidade de Sao Paulo (USP), Sao Paulo. A voucher specimen (CFCR n° 4680) was deposited at the Herbarium of the IB-USP.

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Animals Male Swiss albino mice (25-35 g) from the Central Animal House of the Universidade Estadual Julio de Mesquita Filho (UNESPlBotucatu) were used. The animals were fed a certified Nuvilab CR-a® (Nuvital) diet with free access to tap water under standard conditions of 12 h dark-12 h light and temperature (22 ± 1%). Fasting was used prior to all assays because standard drugs and extract were always administered either orally (by gavage) or by intraduodenal route. The protocols followed the recommendations of the Canadian Council on Animal Care (Olfert et al., 1993).

Drugs and Chemicals Hydrochloric acid (Nuclear, Brazil), cimetidine, atropine (Sigma Chemical Co., St Louis, MO, USA), piroxicam (Hexal, Brazil) and lansoprazole (Medley, Brazil) were used in this study. Extract was dissolved in NaCI solution 0.9% (vehicle). All substances were prepared immediately before use and the reagents used were of analytical grade.

Acute Toxicity The acute toxicity studies of L. flavescens were performed in mice. In this assay, increasing doses ofMeOH extract were orally administered to groups of ten animals for each dose after a 6 h fast. Animals receiving the vehicle served as control. The signs and symptoms associated with the MeOH administration were observed at 0, 30, 60, 120, 180 and 240 min. after and then once a day for the next 14 days. At the end of the period the number of survivors was recorded. The acute toxicological effect was also estimated (Souza Brito, 1995).

Antiulcerogenic Effect HClIethanol-induced ulcer: The antiulcerogenic activity obtained from L. flavescens was studied in HClIethanol-induced gastric ulcer (Mizui & Doteuchi, 1983). Mice were divided into groups of 7-8 animals, which fasted 24 h prior to receiving an oral dose of the vehicle, saline (10 mU kg), lansoprazole (30 mg/kg) or L. flavescens (250, 500 or 1000 mg/kg). After 50 min. all groups were orally treated with 0.2 mL of a 0.3 M HClI 60% ethanol solution (HClIethanol) for gastric-ulcer induction. Animals were killed 1 h after the administration of HClIethanol, and the stomachs excised and inflated by saline injection (2 mL). The extent of the lesions was measured using ulcerative lesion index (ULl) and pH of gastric juice determined. This index was expressed as the sum of all lesions (Szelenyi & Thiemer, 1978).

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Non-Steroidal Antiinflammatory Drug (NSAlD)-Induced Gastric mcers In this model (Puscas et al., 1997), gastric ulcer was induced using piroxicam (30 mg/kg, s.c.) administered to mice. The MeOH extract from L. flavescens (250, 500 or 1000 mg/kg), cimetidine (100 mg/kg) or saline were administered orally 30 min before the induction of gastric ulcer. The animals were killed 4 h after treatment with the ulcerogenic agent and gastric damage was determined as described above.

Hypothermic Restraint Stress mcer Mter fasting, the mice received an oral administration of MeOH extract from L. flavescens (250, 500 or 1000 mg/kg) , cimetidine (100 mg/kg) or saline (10 mUkg). 1 h after treatment, mice were immobilized in a restraint cage at 4°C for 4 h to induce gastric ulcer. The animals were killed and gastric lesions were determined as described above (Levine, 1971).

Determination of Gastric Secretion All groups of mice fasted for 24 h, with free access to water. Immediately after pylorus ligature, MeOH extract from L. flavescens (250, 500 or 1000 mg/kg) , cimetidine (100 mg/kg) as positive control, or the vehicle was administered intraduodenally. The animals were killed 4 h later and the abdomen was opened and another ligature placed around the esophagus close to the diaphragm. The stomachs were removed and the gastric content collected to determine the pH values and total amount of gastric-juice acid. Distilled water was added and the resultant solution centrifuged at 2500 G for 10 min. Total acid in the gastric secretion was determined in the supernatant volume by titration to pH 7.0 with 0.01 N NaOH (Shay et al.,1945).

Anti-Helicobacter pylori Activity The MeOH of L. flavescens was tested to detect anti-Helicobacter pylori activity (Hachem et al., 1996). The stain of Helicobacter pylori (ATCC 43504) was isolated from patients with duodenal ulcer disease. Frozen H. pylori isolate was thawed and grown on 5% sheep blood agar plates for 3 to 4 days at 37°C in 10% CO 2 and 98% humidity. Each plate was swabbed with a sterile cotton-tipped applicator and the cells were suspended in sterile saline to obtain turbidity equivalent to a 2.0 McFarland standard. MullerHinton broth containing 10% horse serum was added to all wells of a 96well microtiter plate (Corning-USA). Each well was incubated with H. pylori at a final concentration of 1 x 105 CFU/mL. The plates were incubated for 5 days in a microaerobic atmosphere at 37°C. Following incubation, the plates were examined visually and spectrophotometrically and the lowest concentration showing complete inhibition of growth was recorded as the MIC (minimum inhibitory concentrations). Staphylococcus aureus ATCC

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25923 and E.coli ATCC 25922 were used as control organisms for clarithromycin and ampicillin, respectively. The results were considered valid only when the MIC values for the control organisms were within the ranges established by the National Committee for Clinical Laboratory Standards (NCCLS).

Determination ofActivity on Intestinal Motility Mice submitted to a 6 h fast were treated orally with the vehicle (saline, p.o.), atropine (1 mg/kg, p.o.), MeOH (250, 500 or 1000 mg/kg, p.o.). 30 min after the treatments, all animals received 10% activated charcoal (0.1 mU 10 g, p.o.) and were killed 30 min later. The distance traveled by activated charcoal up to the last portion of small intestine was determined (Stickney & Northup, 1959). The results were expressed as percentage of the total length of the small intestine traveled by this marker.

Antioxidant Activity The antioxidant activity was evaluated through the methodology of lipidic peroxidation with membrane of brain from Wi star rats (Stocks, 1974), with a serial dilution of the methanolic extract of L. fiavescens (1,14; 2, 27; 4, 55; 9, 09; 18, 18 e 36, 36 )lg/mL) with the purpose to obtain IC 50 (inhibitory concentration of 50% of the oxidation) through the production of malonidialdehide (MDA) (Fee & Teitelbaum, 1972).

Statistical Analysis Results were expressed as the mean ± S.E.M. Statistical significance was determined by one-way analysis of variance followed by Dunnett's test, with the level of significance set at p<0.05.

Extraction and Isolation The scapes ofL. flavescens (500 g) were separated from the capitula, powdered and successively extracted with chloroform, methanol and 80% methanol (1 week for each solvent). The solvents were evaporated in a vacuum yielding black syrups. A portion (2.0 g) of the ethanol extract of L. flavescens was submitted to CC on sephadex LH-20 (100 x 5 cm), with MeOH as eluant. Fractions (8 mL) were collected and checked by TLC on silica gel in Si gel plates, n-BuOH-AcOH-H20 (20:5:8, v/v/v)]. Fractions 90-100 (40 mg) were purified by repeated CC on polyvinylpolypyrrolidone (Sigma) eluted with MeOH yielding compounds 1 (7.0 mg) and 2 (9.0 mg). Fractions 65-73 (69.8 mg) were further purified by HPLC using MeOH:H 20 (1:1, v/v) as eluant to afford 3 (4.2 mg, RT = 8 min), and 4 (10.8 mg, RT = 11 min). Fractions 47-51 (12.0 mg) using MeOH:H 20 (2:3, v/v) as eluant gave compound 5 (6.5 mg, RT = 18 min). Fractions 40-46 using MeOH:H20 (2:3, v/v) as eluant afforded compound 6 (8.5 mg, RT = 9 min) and 7 (5.8 mg, ~ = 16 min.). Fractions 8283 contained pure 8 (13.3 mg). Substances (Fig 1) were identified by NMR (nuclear magnetic resonance, as 13C-NMR, IH-NMR), spectra and by

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comparison with previous data reported in the literature (Santos et al., 2001; Agrawal, 1989; Harborne, 1996).

OR

OR 1,2,4-8 Compounds

Reference(s)

Name

Rl

R2

R3

1 2 4

H H OH

H H glc

OH H OH

5

glc

CH3

OH

6

CH3

glc

OCH 3

7

glc

H

H

8

OH

H

OH

(Agrawal, Luteolin Apigenin 1989; Harborne, 3',4' ,5,6-tetrahydroxy-71996) methoxy-flavone 3',4',5-triihydroxy-7-metoxy6-C-glucopyranosylflavone 4',5-dihydroxy-3',6-dimethoxy7-O-~-D _glucopyranosylflavone 3',4',5, 7-tetrahydroxy-6-Cglucopyranosylflavone 3',4',5,6,7,8-hexahydroxyflavone

COOCH 3 0

HO

OH

OH OH

3- 8-carboxy-methyl-l,3,5,6-tetrahydroxyxanthone (Santos et al., 2001). Fig 1. Flavonoids and xanthones isolated from the scapes of Leiothrix flavescens

Experimental Section NMR spectra in DMSO-d 6 were obtained using a Varian INOVA 500 spectrometer operating at 500 MHz for IH and 150 MHz for 13C. HPLC separations were achieved on a Chance HPLC System equipped with a R401 refractive index detector and with a phenomenex Luna reversed phase C-18 columns (250 x 10 mm x 10 mm) and Rheodyne injector. TLC were performed on silica gel SiF254 (Merck) eluted with CHCI 3:MeOH:H2 0 (80:18:2, v/v/v) and n-BuOH:HAc:H 2 0 (65:35:25, v/v/v), the plates were visualized using UV light (254 and 365 nm).

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HPLC-UV-PDA Analysis The chromatography profile of the methanolic extract of L. flavescens was established with a liquid chromatograph equipped with a PU-2089 quaternary solvent pump, a MD-2010 photo diode array detector (Jasco, Tokyo, Japan) and a Rheodyne 7725 sample injector with a 20 1I1 sample loop (Rheodyne, Cotati, CA, USA). The analytical column was a Phenomenex Luna RP18 (250 x 4.6 mm lD. x 5 lIm, equipped with a Phenomenex security guard 4.0 x 2.0 mm, Torrance, CA, USA). The mobile phase composition used was: water (eluent A) and acetonitrile (eluent B), both with 0.05% ofTFA. The gradient program was as follows: 15-22% B (25 min.), 22-40% B (15 min.), 40-78% B (5 min.), 78-100% B (5 min.) and 100% B isocratic (5 min.). Total run time was 55 min. The flow rate of the mobile phase was 1.0 mUmin. EZChrom Elite Data System software was used for both the operation of detector and for data processing. The methanolic extract (2 mg) was dissolved in 2 mL methanol, filtered through a 0.45 11m membrane PTFE filter (Millex), resuspended in 3 mL of water and 20 I!l submitted to the analysis by HPLC. The HPLC profile of the methanolic extract prepared from scape of L. flavescens was presented in Fig 2. r---

jA

500

1

,]j E

!

Minutes

r~~'~~~\?'\J ,~!f/~\ .. j "'Ie

:lOt ____ ~~______ 2tO

220

a... :no

2M

)DO

m

:MIl

HO

310....

,..10

,I ___ ~_~___ 2DI

no

240

HI

280

_

321

,...

3M

310....

"E ~ 0 __ ~ __. ___ ~ _ no

Z20

241

2M

210

)DO

m

)40

310

* ....

Fig 2. (A) HPLC fingerprint profile of crude methanolic extract from scape ofL. flavescens. Retention times of major peaks are indicated in chromatogram. B) The regions of xanthones are shown as insets. Representative UV for the classes of compounds found in the methanolic extract is shown in the lower part ofthe chromatogram. C) Flavones derivatives, D) Cafeoyl quinic derivatives and E) xanthones derivatives

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RESULTS AND DISCUSSION Biological experiments may be designed with the objective of determining the dose of a chemical required to produce any specific effect. The measured effect need not be death of the animal but may be any type of biologic effect that can be quantified. So as part of the pharmacological evaluation of MeOH extract of L. flavescens, we investigated the acute toxicity in mice. A single oral administration of MeOH extract (5,000 mglkg) did not produce any sign or symptom of toxicity in the treated animals. After 14 days of extract administration, no animal died and no significant changes in daily body or organ weight were observed. There were no significant alterations in water or food consumption. In autopsy, no significant change or lesion was observed in the viscera of each animal. Loomis & Hayes, (1996) described the classification of some chemicals agents with categories of toxicity and chemicals may be categorization by practically nontoxic at dose of 5 glkg. Thus, this result indicates that the MeOH extract from E. ligulatum has no acute toxicological effect when administered orally. These results guarantee the continuance from the pharmacological studies from this specie. For this reason, a 5-fold lower dose (1000 mglkg) was used as the maximum doses in all experiment to determine the general profile of antiulcer effect of extract from L. flavescens scapes. We evaluated the preventive effect of three oral doses of 250, 500 or 1000 mglkg of the MeOH extract obtained from L. flavescens administered to mice using different standard experimental models in order to verify their action on the gastrointestinal tract. This approach allows a general profile to be established for the gastroprotective activity from the vegetal extract for a future investigation of its action mechanism. In the HClIEtOH-induced gastric ulcers, the lesions were characterized by multiple-hemorrhage red bands of different sizes along the long axis of the glandular stomach. As shown in Fig 3, all doses of the MeOH extract showed significant antiulcer activity. The above results observed with MeOH extract (250, 500 or 1000 mg/kg) demonstrated significant inhibition of ulcerative lesion by 51% (24 ± 4.1 mm), 76% (12 ± 4.1 mm) and 87% (6.2 ± 1.9 mm), respectively, compared to the control value (49 ± 7.7 mm). The ability of the gastric mucosa to resist to injury caused by endogenous secretions (acid, pepsin and bile) and by exogenous irritants (e.g. alcohol) can be attributed to a number of factors that have been collectively referred to as mucosal defense (Kinoshita et al., 1995). The formation of gastric mucosal lesions by necrotizing agents such as HCl and EtOH has been reported to involve the depression of these gastric defensive mechanisms and also promote stasis in gastric blood flow that contributes to the development of the hemorrhagic and necrotic aspects of tissue injury (Morimoto et al., 1991).

60

• vehicle D positive control

50

~

40

MeOH 250 mg/kg

I'TI MeOH 500mg/kg 30

lID MeOH 1000 mg/kg

20 10 0 HCl/ethanol

piroxicam

Stress

Fig 3. Effects of oral treatments with MeOH extract from L. fZavescens on ulcerative lesion index (ULI) in gastric lesions induced by HClIethanol, NSAID (piroxicam) and hypothermic-restraint-stress in mice Data are presented as mean ± S.E.M. ANOVA: F(4,32) = 82.8 (p<0.05) for pH-HClIethanol; F(4,32) = 8.88 (p<0.05) for ULI-HClIethanol; F(4,44) = 4.85 for pH-NSAIDS (p<0.05); F I4,44) = 6.27 for ULI-NSAIDS (p<0.05); F I4 ,25) = 0.74 (p>0.05) for pH-stress model and F(4,25) = 6.45 (p<0.05) for ULI-stress model. Dunnett's test: * p<0.05, **p
t>:> -.1

<:.:>

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Gastric lesions induced by ethanol are not inhibited by antisecretory agents such as cimetidine, but are inhibited by agents that enhance mucosal defensive factors such as prostaglandins (Garner, 1992). These results shown that the MeOH extract of L. flavescens probably has an antiulcerogenic effect related to the cytoprotective action since this extract protected against the necrotizing action of ethanol. Nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin®, piroxicam and indomethacin remain among the most commonly used pharmacological agents (Garner, 1992). However, these classes of substances cause gastrointestinal ulceration. This effect is correlated to the ability of these agents to suppress the prostaglandin synthesis (Wallace, 2001). Fig 3, shows the antiulcerogenic effect ofMeOH extract in the NSAIDinduced lesion model. Significant reductions in the damage to gastric mucosa (p<0.05) of 41% (250 mglkg) , 57% (500 mglkg) and 72% (1000 mg! kg), respectively, were observed with the use of the MeOH extract when compared to the control value. The increased synthesis of mucous and/or prostaglandins can be the probable cytoprotective mechanism involved with this activity (Guth et al., 1984). Stress is the major causes of gastric disturbances in modern society. Gastric stress ulceration is probably mediated by histamine release with enhanced acid secretion and reduced mucous production. Moreover, stressinduced ulcer can be prevented partially or entirely by vagotomy. Vagal over-activity has been suggested to be the principal factor in stress-induced ulceration (Singh & Majumdar, 1999). In the gastric ulcer induced by hypothermic-restraint stress (Fig 3), the MeOH extract also showed significant gastroprotection (p<0.05); and the gastric-lesion inhibition for the extract were 60% (500 mglkg) and 70% (1000 mglkg) respectively, when compared with the vehicle. Ares et al. (1995) identified gastroprotective properties in the flavone class and phytochemical analyses of L. flavescens shown that this species produces mainly flavones that certain contributed to gastroprotective action of this plant (Silva et al., 2007). In the next step, we investigated the biochemical alterations promoted by L. flavescens extract in gastric-juice parameters. The MeOH extract was administered intraduodenally after submitting the mice to pylorus ligature (Table 1). Ligation of the pylorus for 4 h produced accumulation of gastric juice and lesion in gastric mucosa, whereas the intraduodenal route showed systemic action for all of the treatments. As shown in Table 1, the MeOH extract significantly decreased the gastricacid secretion (p<0.05) and promoted reduced acid output (p
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arthrotrichus were observed only by flavonoids rich fraction. This results obtained with MeOH extract of L. flavescens as anti secretory agents is important as prophylaxis of ulcer gastric and gastric carcinogenesis. But gastric carcinogenesis is a complex, multistep and multifactorial event, in which the role of Helicobacter pylori infection has been established. We also evaluated the anti-Helicobacter pylori action of MeOH extract of L. flavescens. In the course ofthe study the MeOH extract was found to present an important antibacterial activity against the standard strain (ATCC 43504). The results showed that MIC of MeOH against H.pylori was 125 Ilg/mL. The literature reported that MIC<500 Ilg/mL is considered interesting for vegetal extracts (Gadhi et al., 2001; Hachem et al., 1996). We conclude that the MeOH has an excellent antimicrobial action against one of the most important factors that cause gastric ulceration. Table 1. Effects of MeOH extract from L. flavescens administered intraduodenally (i.d.) on the biochemical parameters of gastric juice obtained from pylorusligature mice Treatment group (p.o.) Control Cimetidine L. flavescens

Dose (mg/kg)

100 250 500 1000

N

9 5 6 6 6

pH (unit) 2.78 3.75 4.00 3.67 3.50

± ± ± ± ±

0.40 0.75 0.45 0.76 0.62

Gastric juice volume (mU4h) 1.55 1.12 1.08 0.94 0.72

0.13 0.11 0.18 0.17 * ± 0.11 ** ± ± ± ±

Acid output (mEqlmU4h) 25.8 15.7 18.3 3.25 5.00

± ± ± ± ±

2.29 3.71 * 2.33 0.48 ** 0.91 **

The results are the mean±S.E.M. N: number of mice used. ANOVA, F(4.27) = 0.80 (p>0.05) for pH; F(4.27) = 5.17 for gastric juice volume (p>0.05); F(4.27) = 18.4 (p<0.05) for [H+]. Dunnett's Test: * p<0.05; ** p
Farinati et al. (2008) shown that among the pathways relevant to gastric carcinogenesis and correlated with H. pylori infection, the production of reactive oxygen species may be quite important. The eradication of H. pylori to prevent the production of reactive oxygen species and accumulation of oxidative DNA damage are strategic point to prevent gastric ulcer and gastric carcinogenesis. The reactive oxygen species (ROS) generate by metabolism of arachidonic acid, platelets, macrophages and smooth muscle cells contribute to gastric mucosal damage (Repetto & Llesuy, 2002). MeOH extract from L. flavescens has an antioxidant activity in vitro with IC 50 of 21.55 ± 0.02 Ilg/mL. This value is considered a good antioxidant activity when it comes from a crude extract and thus justifying the anti ulcerogenic activity observed in other models. We also observed that MeOH extract from L. flavescens was able to increase the propulsion of charcoal meal in a dose dependent manner (Table 2). The velocity of intestinal travel is one of the factors that affected the intensity of luminal absorption and regulated the biodisponsibility of the

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drug administered orally. Thus gut transit test with activated charcoal was carried out to determine the effect of MeOH from L. flavescens on intestinal motility. This result indicated that MeOH extract from L. flavescens also has a laxative property. There are few laxative products related to intestinal peristalsis that have been in use for decades. Many of these products in use today are of plant origin (Fintelmann, 1991). Although laxative effects have been described for some antiulcer drugs (Motiva et al., 1996; Bobrove, 1990), such effects of MeOH extract is being currently investigated by our group. Table 2. Effects of MeOH extract from L. flavescens on intestinal transport, induced by charcoal, in mice (n=71 Treatment group (p.o.) Control Atropine L. flavescens

Dose (mglkg) 5 250 500 1000

Charcoal transport (arcsine) 62.8 40.2 77.0 79.8 83.5

± ± ± ± ±

12.5 16.4# 5.75 5.30 * 7.58 **

The results are the mean ± S.E.M. ANOVA, Dunnett's Test: # p
The phytochemical analyses carried out in this work led to the isolation of xanthones and flavones in the MeOH extract of scapes from L. flavescens (Figs 1, 2). The literature reveals the gastroprotective action of various species containing xanthones and flavones (Gonzalez & Di Stasi, 2002; La Casa et al., 2000; Sartori et al., 1999; Ares et al., 1995). The flavones isolated from L. flavescens are mostly derived from luteoline which presents the catechol nucleus at ring B. Such compounds have been correlated with the antiulcerogenic activity of many plant extracts since they possess antioxidant action (La Casa et al., 2000). Moreover, the isolated xanthone 3 also displays ortho-hydroxyl groups at position 5, 6 of the A ring, thus providing another system with a catechol nucleus. Besides the presence ofxanthones and flavonoids in the MeOH extract of L. flavescens, this extract also has potential as a new alternative in the management of gastric ulcers by the presence of luteolin that has a proven effect against Helicobacter pylori (Chung et al., 2001). In light of the fact that H. pylori is considered the most important risk factor in development of duodenal and gastric ulcers, this study with MeOH extract from L. flavescens as antiulcerogenic agent may contribute an excellent pathway for treating the universal problem of this gastric pathology. In conclusion, our study evidenced the gastroprotective factor of the MeOH extract obtained from L. flavescens, with antioxidant, anti secretory and gastroprotective activities. The phytochemical investigation of the

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277

MeOH extract led to the isolation of flavones and xanthones with catechol nucleus in their structures, which can be correlated with antioxidant activity.

ACKNOWLEDGEMENTS We acknowledge the financial support ofFAPESP, CNPq and Fundunesp.

REFERENCES Agrawal, P.K. (1989). Carbon 13 NMR of flavonoids. Elsevier: Amsterdam. Ares, J.J., Outt, P.E., Randall, J.L., Murray, P.D., Weisshaar, P.S., O'Brien, L.M., Ems, B.L., Kakodkar, S.V., KeIrn, G.R and Kershaw, W.C. (1995). Synthesis and biological evaluation of substituted flavones as gastroprotective agents. Journal of Medicinal Chemistry, 25: 4937-4943. Batista, L.M., de Almeida, AB., de Pietro Magri, L., Toma, W., Calvo, T.R, Vilegas, W. and Souza Brito, AR (2004). Gastric antiulcer activity of Syngonanthus arthrotrichus Silveira. Biological & Pharmaceutical Bulletin, 27: 328-332. Bobrove, AM. (1990). Misoprostol, diarrhoea, and psyllium mucilloid. Annual International Medicine 23: 112-386. Chung, J.G., Hsia, T.C., Kuo, H.M., Li, Y.C., Lee, Y.M., Lin, S.S. and Hung, C.F. (2001). Inhibitory actions of lute olin on the growth and aryl amine Nacetyl transferase activity in strains of Helicobacter pylori from ulcer patients. Toxicol In Vitro, 15: 191-198. Coelho, RG., Batista, L.M., Dos Santos, L.C., Brito, A.RM.S. and Vilegas, W. (2006). Pythochemical study and antiulcerogenic activity of Syngonanthus b~sulcatus (Eriocaulaceae). Brazilian Journal of Pharmaceutical Science, 42: 413-417. Di Stasi, L.C., Camuesco, D., Nieto, A., Vilegas, W., Zarzuelo, A and Galez, J. (2004). Intestinal anti-inflammatory activity of paepalantine, an isocoumarin isolated from the capitula of Paepalanthus bromelioides, in the trinitrobenzenesulphonic acid model of rat colitis. Planta Medica, 70: 315-320. Dokkedal, A.L. and Salatino, A (1992). Flavonoids of Brazilian species of Leiothrix (Eriocaulaceae). Biochemistry Systematic and Ecology, 20: 31-32. Farinati, F., Cardin, R, Cassaro, M., Bortolami, M., Nitti, D., Tieppo, C., Zaninotto, G. and Rugge, M. (2008). Helicobacter pylori, inflammation, oxidative damage and gastric cancer: a morphological, biological and molecular pathway. European Journal Cancer Prevention, 3: 195-200. Fee, J.A and Teitelbaum, H.D. (1972). Evidence that superoxide dismutase plays a role in protecting red blood cells against peroxidative hemolysis. Bwchemistry Biophysic Research Communication, 49: 150-158. Fintelmann, V. (1991). Modern phytotherapy and its uses in gastrointestinal conditions. Planta Medica, 57: 48-52. Gadhi, C.A, Benharref, A, Jana, M. and Lozniewski, A (2001). Anti- Hehcobacter pylori activity of Aristolochia paucinervis pomel extracts. Journal of Ethnopharmacology, 75: 203-205. Garner, A. (1992). Adaptation in the pharmaceutical industry with particular reference to gastrointestinal drugs and diseases. Scandinavwn Journal of Gastroenterology, 27: 83-89. Giulietti, A.M., Amaral, E.C. and Biittrich, V. (1994). Phylogenetic analysis of inter and infrageneric relationships of Leiothrix Ruhl. (Eriocaulaceae). Kew Bulletin, 50: 5571. Giulietti, A.M., Pirani, J.R. and Menezes, N.L. (1998). Estudos em sempre-vivas: importancia economica do extrativismo em Minas Gerais, Brasil. Acta Botanica Brasil, 1: 179-183. Gonzalez, F.G. and Di Stasi, LC. (2002). Anti-ulcerogenic and analgesic activities ofthe leaves of W~lbrandw ebracteata in mice. Phytomedicme, 9: 125-134.

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Guth, P.H., Paulsen, G. and Nagata, H. (1984). Histologic and microcirculatory changes in alcohol-induced gastric lesions in the rat: effect of prostaglandin cytoprotection. Gastroenterology, 87: 1083-1090. Hachem, C.Y., Clarridge, J.E., Reddy, R., Flamm, R., Evans, D.G., Tanaka, S.K. and Graham, D.Y. (1996). Antimicrobial susceptibility testing of Helicobacter pylori. Diagnostic Microbiology Infection Disease, 41: 24-37. Harborne, J.B. (1996). The flavonoids: advances in research since 1986. Chapman & Hall: New York. Kinoshita, M., Tsunehisa, N. and Tamaki, H. (1995). Effect of a combination of ecabet sodium and cimetidine on experimentally-induced gastric-lesions and gastric-mucosal resistance to ulcerogenic agents in rats. Biological Pharmaceutical Bulletin, 18: 223-226. La Casa, C., Villegas, 1., Alarcon de la Lastra, C., Motilva, V. and Martin Calero, M.J. (2000). Evidence for protective and antioxidant properties of rutin, a natural flavone, against ethanol induced gastric lesions. Journal of Ethnopharmacology, 71: 45-53. Levine, R.J. (1971). Peptic Ulcer. Munksgard, Copenhagen, pp. 92-97. Loomis, T.A. and Hayes, A.W. (1996), Essentials of Toxicology (4th edn). Academic Press Limited: London. pp. 33-46. Mizui, T. and Doteuchi, M. (1983). Effect ofpolyamines on acidified ethanol- induced gastric lesions in rats. Japanese Journal of Pharmacology, 33: 939-945. Morimoto, Y., Shimohara, K., Oshima, S. and Sukamoto, T. (1991). Effects of the new antiulcer agent KB-5492 on experimental gastric mucosal lesions and gastric mucosal defensive factors, as compared to those of terprenone and cimetidine. Japanese Journal Pharmacology, 57: 595-605. Motilva, V., Lopez, A., Martin, M.J., La Casa, C. and Alarcon de la Lastra, C. (1996). Cytoprotective activity of cisapride on experimental gastric mucosal lesions induced by ethanol. Role of endogenous prostaglandins. Prostaglandins, 52: 63-74. Olfert, E.D., Cross, B.M. and McWilliam, A.A. (1993). Guide to the Care and Use of Experimental Animals. Canadian Council on Animal Care Co: Ontario pp. 1-213. Puscas, 1., Puscas, C., Coltau, M., Pas~a, R., Torres, J., Marquez, M., Herrero, E., Fillat, O. and Ortiz, J.A. (1997). Comparative study of the safety and efficacy of ebrotidine versus ranitidine and placebo in the prevention of piroxicam-induced gastroduodenal lesions. Arzneimittelforschung, 47: 568-572. Repetto, M.G. and Llesuy, S.F. (2002). Antioxidant properties of natural compounds used in popular medicine for gastric ulcers. Brazilwn Journal of Medicinal Biological Research, 35: 523-534. Salatino, A., Salatino, M.L.F. and Giulietti, A.M. (1990). Contents of soluble phenolic compounds of capitula of Eriocaulaceae. Quimica Nova, 13: 289-292. Santos, L.C., Piacente, S., De Ricardis, F., Eletto, A.M., Pizza, C. and Vilegas, W. (2001). Xanthones and flavonoids from Leiothrix curvifolia and Leiothrix {lavescens. Phytochemistry, 56: 853-856. Sartori, N.T., Canepelle, D., de Sousa, P.T. Jr. and Martins, D.T. (1999). Gastroprotective effect from Calophyllum brasiliense Camb. bark on experimental gastric lesions in rats and mice. Journal of Ethnopharmacology, 67: 149-156. Shay, H., Komarov, S.A., Fels, S.S., Meranze, D., Gruestein, M. and Siplet, H. (1945). A simple method for the uniform production of gastric ulceration in the rat. Gastroenterology, 5: 43-6l. Silva, M.A., Oliveira, A.P., Sannomiya, M., Sano, P.T., Varanda, E.A., Vilegas, W. and Santos, L.C. (2007). Flavonoids and a naphthopyranone from Eriocaulon ligulatum and their mutagenic activity. Chemical and Pharmaceutical Bulletin, 55: 1635-1639. Singh, S. and Majumdar, D.K. (1999). Evaluation of the gastric antiulcer activity of fixed oil of Ocimum sanctum (Holy Basil). Journal of Ethnopharmacology, 65: 13-19. Souza Brito, A.R.M. (1995). Manual de Ensaios Toxicologicos in vivo. Editora da UNICAMP, Campinas, Sao Paulo. Stickney, J.C. and Northup, D.W. (1959) Effect of gastric emptying upon propulsive motility of small intestine in rat. Proceed Society Experimental Biological Medicine, 10: 101-582.

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Stocks, J., Gutteridge, J.M., Sharp, R.J. and Dormandy, T.L. (1974). Assay using brain homogenate for measuring the antioxidant activity of biological fluids. Clinical Science Molecular Medicine, 47: 215-222. Szelenyi, I. and Thiemer, K. (1978). Distention ulcer as a model for testing of drugs for ulcerogenic side effects. Archieve Toxicolology, 41: 99-105. Wallace, J.L. (2001). Mechanisms of protection and healing: Current knowledge and future research. Amencan Journal Medicine, 110: 19-22.

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14 Evaluation of the Immunity Potency and Security of Inactivated Oil-Adjuvant Vaccine against Chlamydiosis in Dairy Cattle Qru, C.l,*, ZHOU, J.l, CAO, X.l

AND

LIN, G.l

ABSTRACT The inactivated oil-adjuvant vaccine against chlamydiosis in dairy cattle was successfully developed using Chlamydia psittaci SX5 field strain with strong and stable virulence in China. The minimal effective dosages for adult dairy cow and calf are 3 mL and 1.5 mL, respectively, and the clinically used dosages are 4-5 mL (adult cattle) and 2 mL (calves), respectively. The average protection rate reaches 94.4% (ranging from 88.9% to 100%) in the vaccines potency tests. The immunity persistent periods of the vaccine is ten months (adult cattle) and six months (calf). The vaccines conserved for 12 months at 4°C has effects same as the new one. The safe tests for cattle of various ages and small areas of field trials (1216 cattle were vaccinated) confirmed safety of the vaccine is reliable and there were not obviously abnormal changes in appetite, milk production and the site of vaccination in the vaccinated cattle herds. Key words : Chlamydiosis in dairy cattle, inactivated vaccine, protection rate, safety

INTRODUCTION Chlamydiae are widely distributed throughout the world, causing various forms of disease in animals and humans. Several species, particularly Chlamydophila (Cp.) psittaci and Cpo abortus, are known to be transmissible 1. State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research

*

Institute, CAAS, Lanzhou Gansu 730 046, China. Corresponding author: E-mail: [email protected]

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from animals to humans, causing significant zoonotic infections (Gnutov et al., 1980; Stepanek et al., 1983; Sachse et al., 2002). Chlamydiae is strictly intracellular microorganism, which have unique biphasic lifestyle, namely infective extracellular phage and parasitic intracellular phage, which renders the respective diseases difficult to control. This is compounded by the specialist growth conditions for the organisms and the lack of a genetic based system for the transformation of chlamydiae, both of which have hampered research on these pathogens. To escape the host immune response these bacteria are capable of transforming into persistent stages of development characterised by a distinct antigenic profile. Bovine chlamydial abortion is one of all serious diseases induced by Chlamydia, which may result in large loss in dairy industry (Daniel et al., 1993; Kaltenboeck et al., 2005). The disease is named as epizootic bovine abortion caused by Cp abortus, and characterized by abortion, premature birth, still birth or weak birth with wide prevalence (Martel et al., 1983; Martinov, 1984). The disease often aggrieves virgin cows, heifers or cows from nonepidemic areas and results in 25% to 75% of affected pregnant cows to abort and infertility (Bowen et al., 1978). The causative agents may induce uteritis and infertility in cows with bad curative effect despite drugs (Veznik et al., 1996), conjunctivitis (Otter et al., 2003), mastitis and polyarthritis (Martinov et al., 1981), etc. In a regional survey in Germany, Cpo psittacilCp. abortus were detected at a rate of up to 100% in affected herds. Economic losses as a consequence of a drop in milk production and milk quality, as well as abortions and reduced fertility rates were estimated to be Euro 40,000 per year at an average farm of 60 dairy cows and 20 heifers. Investigations carried out in Italy have shown that the chlamydial organisms involved in these outbreaks belong to the so-called "noninvasive" strains classified as Cpo pecorum. The same agent was also detected in cases of encephalomyelitis in water buffaloes used for milk production (Qiu, 2003). In 1980's, investigations of bovine chlamydiosis carried out in some provinces in China showed infection rate of chlamydiosis in dairy cows was higher than in yellow cattle or buffalo (Qiu, 2005). In current years, the dairy farming has been developing rapidly and at present has reached above ten million cattle in China. However, the production level of dairy cattle was very low owing to prevalence of infectious diseases (including chlamydiosis) (Qiu et al., 2006) and absence of specific bio-preparations for monitor and control of dairy cattle diseases. So we carried out studies on inactivated vaccine against chlamydiosis in dairy cattle.

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283

MATERIALS AND METHODS The Vaccine Strain The vaccine strain is Chlamydia psittaci SX5 which was isolated from a dairy cow affected with chlamydial abortion on a dairy farm standing at the north of Xi-an city of Shaanxi province locating in the North-western of China, and identified and preserved in our laboratory and its virulence value reached 1O-12ELD5JOA mL (Qiu et al., 2006).

The Virulent Strain Used in the Vaccine Potency Test The virulent strain used in the vaccine potency test is Chlamydia psittaci SX5 with virulence value of 1O-12ELD 5JOA mL. In the potency tests, the calves were challenged at a dosage of 1 mL and adult dairy cattle at dosage of 2 mL by neck vein route (The concentration of the virulent strain SX5 used in the vaccine potency test was 10% of suspension of the strain SX5 chicken embryo yolk sac cultures). And the challenges were carried out at the third week post vaccinations in the potency tests (Li et al., 1995).

The Tested Vaccine The ten batches of inactivated oil-adjuvanted vaccines against chlamydiosis in dairy cattle were prepared for potency test and security test of target animals and the vaccines were composed of inactivated SX5 antigens and Montanide ISA 206 oil adjuvant made in France with 0.2% formaldehyde as inactivator. In these trials, calvies and adult cattle were intramuscularly vaccinated at a doses of 2 mL and 4-5 mL, respectively.

The Vaccine Tests The contents of vaccine evaluation examination included potency test, safety test, immune persistence period test for dairy cattle and conservation time of the vaccine in the laboratory as well as field tests of vaccinations of target animals.

Animals used in the tests: All cattle were purchased from several cattle herds which had been confirmed to be negative for Chlamydia infection by serologic method.

RESULTS The Minimal Effective Dosages for Target Animal The minimal effective dosages of the vaccine for adult cattle and calf were seen in the Tables 1 and 2, respectively.

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Table 1. The minimal effective dosages for adult dairy cow Groups 1 2 3 4 5 6 Control

Vaccine batch no.

Cattle

030312 030317 030327 030312 030317 030327

5 5 5 5 5 5 5

Date 03-03-30 03-03-30 03-03-30 03-03-30 03-03-30 03-03-30 03-03-30

Dose

Challenge

(mL)

(mL)

Protection rate

2.0 2.0 2.0 3.0 3.0 3.0 0

2.0 2.0 2.0 2.0 2.0 2.0 2.0

2/5 2/5 2/5 4/5 5/5 5/5 0/5

Dose

Challenge

(mL)

(mL)

Protection rate

1.0 1.0 1.0 1.5 1.5 1.5 0

1.0 1.0 1.0 1.0 1.0 1.0 1.0

1/5 215 215 5/5 4/5 5/5 0/5

Table 2. The minimal effective dosages for dairy calvies Groups 1 2 3 4 5 6 Control

Vaccine batch no.

Cattle

030312 030317 030327 030312 030317 030327

5 5 5 5 5 5 5

Date 03-03-30 03-03-30 03-03-30 03-03-30 03-03-30 03-03-30 03-03-30

According to the results of minimal effective dosages tests, the minimal effective dosages of the vaccine for adult cattle and calf were 3.0 mL and 1.5 mL, respectively. Clinically, the dosages used for calves and adult cattle were affirmed as 2 mL and 4-5 mL, respectively.

The Vaccine Potency Tests Six batches of the vaccines were used for two potency tests, and in the first potency test, the vaccine protection rate was 8/9, and in the second potency test, the vaccine protection rate was 9/9, with an average protection rate of 94.4%. The detail was seen in the Table 3. Table 3. Potency tests of the vaccine for dairy cattle Groups

Vaccine Cattle batch no.

Dose (mL)

Challenge Smear Isolation Protection (mL) rate(%)

1 2 3 Control 1

030312 030327 030403

3 3 3 3

5.0 5.0 5.0 0

2.0 2.0 2.0 2.0

3/3 3/3 3/3 3/3+

3/3 3/3 2/3 3/3+

3/3 (100.0) 3/3 (100.0) 2/3 (66.7) 0/3 (0)

4 5 6 Control 2

040113 040210 040317

3 3 3 3

5.0 5.0 5.0 0

2.0 2.0 2.0 2.0

3/3 3/3 3/3 3/3+

3/3 3/3 3/3 3/3+

3/3 (100.0) 3/3 (100.0) 3/3 (100.0) 0/3 (0)

Immunity of Inactivated Oil-Adjuvant Vaccine against Chlamydiosis

285

The Safety Tests The security tests of the vaccine would deal with weaned calves (30 to 40 days old), pregnant cows and adult cattle. The details were seen in the Tables 4, 5, 6, respectively. Table 4. Safety tests of the vaccine for dairy calvies Groups 1 2 3 4 5

Vaccine batch no.

Calves

030312 030327 030403 040113 040210

5 5 5 5 5

Dose (mL)

5.0 5.0 5.0 5.0 5.0

Site of vaccination No No No No No

swelling swelling swelling swelling swelling

Appetite

Evaluation

Normal Normal Normal Normal Normal

Safe Safe Safe Safe Safe

Table 5. Safety tests of the vaccine for pregnant dairy cows Groups Vaccine Pregnant Dose (mL) batch no. cows 1 2 3 4 5

030312 030327 030403 040113 040210

5 5 5 5 5

10.0 10.0 10.0 10.0 10.0

Site of vaccination absorded in absorded in absorded in absorded in absorded in

Parturition Evaluation

3 days 2-3 days 2-3 days 2-3 days 2-3 days

Normal Normal Normal Normal Normal

Safe Safe Safe Safe Safe

Table 6. Safety tests of the vaccine for adult dairy cattle Groups 1 2 3 4 5

Vaccine Adult batch no. cows 041210 050103 050114 050120 050220

5 5 5 5 5

Dose (mL)

10.0 10.0 10.0 10.0 10.0

Site of vaccination absorded in 2-3 days absorded in 3 days absorded in 2-3 days absorded in 2-3 days absorded in 3 days

Clinically Evaluation Normal Normal Normal Normal Normal

Safe Safe Safe Safe Safe

The test results of safety of the vaccine for target animals well indicated that after the vaccinations, no tested cattle showed serious manifestations with basically normal appetite and milk crops clinically. And all of the vaccinated pregnant cattle did not aborted within the whole test duration. A few of the tested animals had a high temperature (to 39°e or so) but returned normal in the second day post the vaccinations.

The Immune Persistence Period Test of the Vaccine Nine adult cattle and nine calvies were inoculated with the vaccines and then the sera were continuously sampled from the tested animals for ten months (adult cattle) or six months (calves) for measuring the antibody levels to chlamydiosis. The antibody titer changes were seen in the Fig 1 and 2 in detail.

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286

N

Cl

Qj

;>,

"0

10 8

6

-+- 3mL-group

4

- . - 5mL-group

0

.0

~

2 0 0

..... N

0

CD

0

(j)

0 N

0

L()

0 0 ex:> ..... ..... N

0

<;f"

N

0 ..... N

0 0

(V)

If)

;>,

co

"0

Fig 1. The antibody curves in adult dairy cattle inoculated with various doses of the vaccine within 10 months duration

As showing in the Fig 1, the antibody titers of the both group animals reached 1:16-1:32 on the day 21 post vaccination; the antibody titers of the both group animals were kept in an increasing status and reached 1: 512 on the day-90 post vaccination; the antibody titers of the both group animals were steady in 1: 256 or so within 4 to 8 months after vaccinations; in 9 to 10 months post vaccinations, the antibody level of the 3 mL-group animals began to fall but the antibody level of the 5 mL-group animals was kept in 1: 128-1: 256.

The adult cattle of the 5 mL-group were challenged with virulent strain SX5 10 months after the vaccinations and all inoculated animals were protected and the test results were seen in the Table 7 in detail.

12 10

'ON ... en

~~

~.~

'0'0 0>-

.oE ._
c:C

-+-Group I ---Group II ---.....- Group III

8 6

4

~ Control

«u 0

o

15

30

60

90

120 150 180

Days

Fig 2. The antibody curves in calvies inoculated with the vaccine within 6 months duration

Immunity of Inactivated Oil-Adjuvant Vaccine against Chlamydiosis

287

Table 7. The challenge test in the immunity persistent period of the vaccine for adult cattle Groups 1 2 3 Control

Vaccine Cattle batch no. 030312 030327 030403

3 3 3 3

Dose (mL)

Challenge Smear Isolation Protection (mL) rate(%) 3/3 3/3 3/3 3/3+

2.0 2.0 2.0 2.0

5.0 5.0 5.0 0

3/33/3 3/3 3/3+

3/3 (100.0) 3/3 (100.0) 3/3 (100.0) 0/3 (0)

15 days after the vaccinations, the antibodies (1:64) could be detected from most of the inoculated calves. And 6 months after the vaccination, the antibody titers were kept at higher levels in inoculated calves of the 3 groups and the mean antibody titers were 10 Lg2, 9.3 Lg2 and 10 Lg2, respectively. While all of the calves were challenged, the animals of the vaccinated groups all were protected (3/3, 3/3, 3/3), but no animals of the control group were protected (0/3). The test results were seen in the Table 8 in detaiL The 3/3 calves of the control group showed high body temperature (39.8°C to 40.1°C and the 1/3 calves diarrhoea occurred after challenging. Table 8. The challenge test in the immunity persistent period of the vaccine for calvies Groups

Calvies

Inoculated dose(mL)

Antibody titer 180 days post vaccination

Challenge dose(mL)

Protection rate%

I

3 3 3

2.0 2.0 2.0 3

10Lg2 9.3Lg2 lOLg2 0

1.0 1.0 1.0 1.0

3/3 (100.0) 3/3 (100.0) 3/3 (100.0) 0/3(0)

IT III Control

The conservation time of vaccine The three batches of the vaccine which had been hold for 12 months at 4°C were used to inoculate 9 dairy cattle, each team 3 animals. Challenging on the day 21 post the vaccinations, all of the vaccinated animals were protected but the 3 control animals fell ill showing high body temperature, bad appetite or abortion. The test results were seen in the Table 9. Table 9. Storage period tests of the vaccine Groups 1 2 3 Control

Vaccine Cattle Vaccination batch no. date 030312 030327 030403

3 3 3 3

04-04-05 04-04-05 04-04-05 04-04-05

Dose (mL)

Challenge date

5.0 5.0 5.0 0

04-04-26 04-04-26 04-04-26 04-04-26

Dose Protection (mL) rate% 2.0 2.0 2.0 2.0

3/3 (100.0) 3/3 (100.0) 3/3 (100.0) 0/3(0)

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According to the test results, the quality of the vaccines kept for 12 months at 4°C did not go down. So the conservation time of the vaccine was a year.

The Vaccine Field Test In order to further know effect and safety of the vaccine for dairy cattle in field, the three batches of the vaccine were used in field tests and 1216 dairy cattle were vaccinated at some of the dairy farms in Gansu, Ningxia, Inner Mongolia, Shaanxi, Henan, Guangdong, Zhejiang and Sichuan provinces, China, which proved that the vaccines were effective for preventing and controlling bovine chlamydiosis and very safe. For example, in 2003 year, on a dairy farm with above one thousand cattle, six hundred of which became pregnant in Ningxia province, chlamydiosis took place,which induced above 100 pregnant cows to abort within a month with suffering heavy economic loss. Mterwards, when all animals of the herd were vaccinated with the vaccines, abortions of dairy cows were stopped rapidly and the production of the farm recovered normality also. DISSCUSSION Bovine chlamydial abortion is one of chlamydiosises leading to very severe economic loss in dairy farming in China. Now the natural transmission manner of the disease has been unclear. The affected animals and latently infected hosts carrying the pathogen and showing no clinical manifestations were very important infection origins (Reinhold et al., 2008). It might be possible that the disease was transmitted between cattle and sheep each other (Griffiths et al., 1995). While pregnant cows with chlamydiosis aborted or laboured, a lot of Chlamydia would be discharged out with the amniotic fluids (Wittenbrink et al., 1994) and polluted environment around and infected other susceptible animals via ingestive tracts (Wittenbrink et al., 1993) and respiratory tracts (Ehret et al., 1975). If a breeding bull was infected by Chlamydia psittaci, cow herds copulated with the bull might be infected by Chlamydia- contaminated sperm (Storz et al., 1968; Amin 2003; Kauffold et al., 2007). Investigations revealed that if not controlling, bovine chlamydiosis would be rapidly spread and induce very severe economic loss in cattle farming (Stepanek et al., 1983). Because of extensive occurrences and many infection routes of Chlamydia spp, the complex strategy characterized by vaccination and strong management would be needed for prevention and control of chlamydiosis in cattle (Rodolakis et al., 1987; Rodolakis et al., 1998; DeGraves et al., 2004; Biesenkamp-Uhe et al., 2007). The very pivotal aspect for developing a vaccine is selection of immunogenecity-broad, virulence-steady vaccine strain. In the study, C. psittaci SX5 was confirmed to be virulence-strong and stable by continuous passages in chick embryos, clone and sequence of the MOMP gene and

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potency test of target animal and highly identical in homology of the MOMP genes of prevalence strains from other regions (Qiu et al., 2006). So the SX5 is a more ideal strain for vaccine. According to minimal vaccination dosage test, the dosages used clinically for calvies and adult cattle were 2 mL and 4-5 mL, respectively. The dosages for adult dairy cows would be chosen on the basis of the milk production and individual bodily form size, if the milk production was higher and the individual bodily form size was bigger, the vaccination dose would be 5 mL, otherwise, 4 mL. In the security tests, because two-months-pregnant cows were easily infected with Chlamydia sp the safety of the vaccine to two-monthspregnant cows was examined which showed that the vaccine was much safe for both mother and fetus. So the efficacy of vaccinations for preventing chlamydial infection in dairy cows would be reasonable in a month before or after artificial inseminations. On the basis of prevalence characteristics of bovine chlamydiosis and duration of the vaccine protection, the use procedure of the vaccine: cows will be vaccinated in a month before or after artificial inseminations; calves will be vaccinated in a month after weaning.

ACKNOWLEDGEMENTS This research was supported by The director funds of Lanzhou Veterinary Research Institute (No. 2003-6; No. 2005-5); The national specific fund of dairy industry (No. 2002BA518A04); The national science-technology support plant fund (No. 2006BAD 04A05) and the Gansu province grand specific project fund of biologic technique (No. 0702NKDA037).

REFERENCES Amin, A.S. (2003). Comparison of polymerase chain reaction and cell culture for the detection of Chlamydophila species in the semen of bulls, buffalo-bulls, and rams. Vetennary Journal, 166(1): 86-92. Biesenkamp-Uhe, C., Li, Y., Hehnen, H.R., Sachse, K. and Kaltenboeck, B. (2007). Therapeutic Chlamydophila abortus and C. pecorum vaccination transiently reduces bovine mastitis associated with Chlamydophila infection. Infection and Immunity, 75(2): 870-7. Bowen, RA., Spears, P., Stotz, J. and Deidel, G.E. Jr. (1978). Mechanisms of infertility in genital tract infections due to Chlamydia psittaci transmitted through contaminated semen. The Journal of Infective Diseases, 138(1): 95-8. Daniel, RG., Holliman, A., David, G.P., Kirby, F.D., Simpson, V.R, Cranwell, M.P., Dawson, M. and Griffiths, P.C. (1993). Bovine chlamydiosis in the United Kingdom. The Veterinary Record, 133(14): 351-35. DeGraves, F.J., Kim, T., Jee, J., Schlapp, T., Hehnen, H.R and Kaltenboeck, B. (2004). Reinfection with Chlamydophila abortus by uterine and indirect cohort routes reduces fertility in cattle preexposed to Chlamydoph~la. Infectwn and Immunity, 72(5): 2538-45. Ehret, W.J., Schutte, A.P., Pienaar, J.G. and Henton, M.M. (1975). Chlamydiosis in a beef herd. Journal of the South Afncan Veterinary Association, 46(2): 171-179.

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Gnutov, LN. and Erokhina, S.G. (1980). Clinico-serologic study of humans with diseases epidemiologically related to halproiosis(chlamydiosis) of farm animals. Zhurnal Mikrobiologii, Epidemiologii, IImmunobiologii, (10): 26-31 (in Russian). Griffiths, P.C., Plater, J.M., Martin, T.C., Hughes, S.L., Hughes, KJ., Heminson, R.G. and Dawson, M. (1995). Epizootic bovine abortion in a dairy herd: characterization of a Chlamydia ps!ttac! isolate and antibody response. The British Veterinary Journal, 151(6): 683-93. Kaltenboeck, B., Hehnen, H.R. and Vaglenov, A. (2005). Bovine Chlamydophila spp. infection: do we underestimate the impact on fertility? Veterinary Research Communications, 29(Suppl 1): 1-15. Kauffold, J., Henning, K, Bachmann, R., Hotzel, H. and Melzer, F. (2007). The prevalence of chlamydiae of bulls from six bull studs in Germany. Animal Reproduction Science, 102(12): 111-21. Li, Y., Shuai, Y., Duan, Y., Gao, S., Yang, X.J., Qiu, C., Yang, X.L., Yi, Q., Zheng, L., Chen, Z., Zhou, G., Pang, B., Liu, Q., Wei, Q. and Li, Z. (1995). Development of inactivated vaccine against chlamydial abortion in pigs. Chinese Journal of Veterinary Science and Technology, 25(11): 3-7 (in Chinese). Martinov, S., Popov, G. and Panova, M. (1981). Chlamydial polyarthritis in calves. Veterinarno- Meditsinski Nauki, 18(3): 83-91 (in Bulgarian). Martel, J.L., Perrin, M., Rodolakis, A., Russo, P., Deschanel, J.P. and Garnier, F. (1983). Experimental infection of the pregnant cow with Chlamydia psittaci. Annals of Veterinary Research, 14(2): 117-20 (in French). Martinov, S. (984). Chlamydial infection in herds of cattle with abortions. VeterinarnoMeditsinski Nauki, 21(10): 81-8 (in Bulgarian). Otter, A., Twomey, D.F., Rowe, N.S., Tipp, J.W., McElligott, W.S., Griffiths, P.C. and O'Neill, P. (2003). Suspected chlamydial keratoconjunctivitis in British cattle. The Veterinary Record, 152 (25): 787-8. Qiu, C. (2003). Chlamydiosis in animals and COST study. Infornations on Animal-Veterinary Science and Technology, 19(8): 21-24 (in Chinese). Qiu, C. (2005). Bovine chlamydiosis and its control. China Animal Health, 3: 18-20 (in Chinese). Qiu, C., Zhou, J., Cheng, S. and Cao, X. (2006). Identification and immunogenicity tests of pathogen causing chlamydial abortion in dairy cows. Vetermary Science in China, 36(4): 270-273 (in Chinese). Qiu, C., Hou, Y., Zhou, J., Cao, X. and Zhang, F. (2006). Epidemiological investigation of chlamydiosis in dairy cattle in China. Indwn Journal of Animal Scwnces. 76(12): 985-987. Reinhold, P., Jaeger, J., Liebler-Tenorio, E., Berndt, A., Bachmann, R., Schubert, E., Melzer, F., Elschner, M. and Sachse, K. (2008). Impact of latent infections with Chlamydophila species in young cattle. Veterinary Journal, 175(2): 202-11. Rodolakis, A., Salinas, J. and Papp, J. (1998). Recent advances on ovine chlamydial abortion. Veterinary Research, 29(3-4): 275-288. Rodolakis, A. and Souriau, A. (1987). Vaccination against bovine chlamydial abortion with a temperature- ensitive mutant of Chlamydia psittaci. Annals of Veterinary Research, 8(4): 439-41 (in French). Sachse, K and Grossmann, E. (2002). Chlamydial diseases of domestic animals-zoonotic potential of the agents and diagnostic issue. Deutsche tieriirztliche Wochenschift, 109(4): 142-8 (in German). Stepanek, 0., Jindrichova, J., Horacek, J. and Krpata, V. (1983). Chlamydiosis in cattle and in man: an epidemiologic and serologic study. Journal of Hygiene, Epidemiololy, Microbiology and Immunology, 27(4): 445-59. Storz, J., Carroll, E.J., Ball, L. and Faulkner, L.C. (1968). Isolation of a psittacosis agent (Chlamydia) from semen and epididymis of bulls with seminal vesiculitis syndrome. American Journal of Veterinary Research, 29(3): 549-55. Veznik, Z., Kummer, V., Canderle, J., Maskova, J., Svecova, D., Pospil, L., Diblikova, L and Zraly, Z. (1996). Detection of Chlamydia in inflammation of the cervix and uterus in cows. Veterinarnl Mediclna (Praha), 41(10): 297-304 (in Czech).

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Wittenbrink, M.M., Kirpal, G., Thiele, D., Fische,r D., Krauss, H. and Bisping, W. (1994). Detection of Chlamydia psittaci in vaginal discharge of cows: a necessary enlargement of bacteriologic diagnosis for the etiologic clarification of fertility disorders in the female cow. Journal of Veterinary Med~clne Series B, 41(7-8): 492-503 (in German). Wittenbrink, M.M., Bisping, W., Mrozek, M. and Horchler, H. (1993). Intestinal Chlamydia psittaci infection of cattle: frequency and technical aspects of the cultural detection of the agent. Deutsche tierarztliche Wochenschift, 100(5): 195-8 (in German).

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15 Annona crassiflora Wood Constituents: Antimalarial Efficacy, Larvicidal and Antimicrobial Activity MARy Am; GoNQALVESl, VANESSA CARLA FURTADO MOSQUEIRA2 AND LUCIA PINHEIRO SANTOS PIMENTA 1,*

ABSTRACT Wood of Annona crassiflora was phytochemically evaluated and three annonaceous acetogenins, annofolin (1) ACM5 (2) and ACM6 (3), the alkaloids aterospermidine (4) and liriodenine (5), the steroids f3-sitosterol (6) and stigmasterol (7), and fatty acids were isolated. Their structures were elucidated by a combination of chemical and spectral methods including MS and NMR experiments. Antimalarial activity, antimicrobial activity in vitro against pathogenic bacteria and brine shrimp lethality of wood extracts of Annona crassiflora were evaluated. The crude extract and fractions were very toxic to brine shrimp (DL 50<30 J.1g/mL). Doses of the extract and fractions of 500 mg/kg and 250 mg/kg caused animal death upon injection in P.berghei-infected mice. Doses of 12.5 mg/kg and 1.25 mg/kg produced 67% suppression of parasitaemia. The alkaloidic fraction showed good results when compared with chloramphenicol against pathogenic bacteria. Key words: Annona crassiflora, Annonaceae, antibacterial activity, antimalarial efficacy, larvicidal activity, Plasmodium berghei

1. Departamento de Quimica - Instituto de Ciencias Exatas - Universidade Federal de

Minas Gerais, Av. Antonio Carlos 6627, BeloHorizonte, MG, Brazil; 31270-901. 2. Departamento de Farmacia- Escola de Farmacia - Universidade Federal de Duro Preto, Duro Preto, MG. * Corresponding author: E-mail: [email protected]

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INTRODUCTION It is estimated that Brazil has about 60-250000 plant species that still are lesser known and under explored. Probably, 40% of them possesses interesting biological activities, constituting source of new useful molecular standards for the development of new plant-derived drugs. Our team has been investigating Annonaceae family for over ten years (Santos et al., 1995; Pimenta et al., 1996; Pimenta et al., 2001; Santos et al., 2006) to find plants with potential activity as antimicrobial, against endemic illnesses such as malaria, and chronic-degenerative diseases as the cancer. Annonaceae family has been source of several structural types of isoquinoline alkaloids and Annonaceous acetogenins. Annonaceous acetogenins are long chain fatty acid derivatives isolated exclusively from plants belonging to the Annonaceae family. These natural products exhibit a broad range of biological properties such as cytotoxic, immunosuppressive, pesticidal, antiparasitic, and antimicrobial activities. Furthermore, its potential to inhibit multiple drug resistant cells have attracted increasing interest in these compounds (Bermejo et al., 2005). Our previous phytochemical study with the seeds of Annona crassiflora has yielded three new and four known annonaceous acetogenins that showed good cytotoxic activity against some human tumoral cells lines (Santos et al., 1995; Pimenta et al., 1996; Pimenta, 1995). Insecticidal activity against Spodoptera frugiperda was also detected from leaves and seeds extracts of Annona crassiflora (Pimenta et al., 2000). The Annonaceous acetogenins had also been determined earlier to possess antiparasitic activities, such as antimalarial in vivo (Rupprecht et al., 1990; Fang et al., 1993). Isoquinoline alkaloids have shown great activity against resistant strains of P. falciparum. (Fisher et al., 2004). Xylopia aromatica bark extract (Annonaceae) was active against Plasmodium falciparum chloroquine resistant strain in vitro but inactive against P. berghei ANKA strain in vivo (Garavito et al., 2006). Considering that malaria is the most prevalent disease among the insectborne individuals and kills around two million people every year, it is very important to look for antimalarial active new drugs. The success of the antimalarial drugs, quinine and artemisinin, from plant's sources, associated to the ethnopharmacological information about the Xylopia aromatica activity in vitro against P. falciparum (Garavito et al., 2006) and also the presence of the acetogenins and isoquinoline alkaloids in this species drove our attention to the evaluation of the antimalarial activity of the wood's crude extracts of A. crassiflora and some fractions from them. The species Annona crassiflora, popularly known as "araticum, cabe~a­ de-negro", or "marolo", is a native tree from Brazilian "cerrado". It ocurrs in the states of Minas Gerais, Bahia, Ceara, Goias, Mato Grosso and Sao Paulo. Its fruit is edible and used as juice, ice-cream, jelly and liqueur. Its pulp is laxative and the seeds are used against diarrhoea and once pulverized they are toxic and reputed as anti-parasitic. It has been used as efficient

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remedy for lice elimination. There are reports of leaves being used as medication for sweat, antirheumatism by oral route. It may also be used externally, as a mouth rinse, in treatment of stomach inflammations, and in neuralgias and headaches (Lorenzi et al., 2002). Prior to our investigation some alkaloids have been isolated from the leaves (Hocquemiller et al., 1982), and cytotoxic annonaceous acetogenins from the seeds (Santos et al., 1994; Santos et al., 1999; Pimenta, 1995) of this plant.

Annona crassiflora is disappearing due to the fire used by local farmers for preparing the field for crops. Neither chemical nor biological studies of this plant's wood are reported in the literature. The present paper reports the fractionation of A. crassiflora wood extract bioguided by Artemia salina lethality test which lead to the isolation of three annonaceous acetogenins, named annofolin, ACM5 and ACM6 (1, 2, 3), the alkaloids aterospermidine (4) and liriodenine (5), the steroids psitosterol (6) and stigmasterol (7), and fatty acids. The antimalarial efficacy and the general toxicity of the extracts and some fractions were evaluated in vivo in the Plasmodium berghei-infected mice following the conventional four-day-test (Peters et al., 1986). Antimicrobial activity was determined in vitro against pathogenic bacteria S. aureus, B. subtilis, E. coli, M. luteus and P. aeruginosa (Takahashi et al., 2006).

MATERIALS AND METHODS Melting points determinations were determined on a Mettler FP5 apparatus and are uncorrected. Optical rotations were taken on a PERKIN ELMER 341 polarimeter. IR spectra were measured on a Perkin Elmer spectrometer version 3.02.1 and Spectrum One-FT-IR Spectrometer Universal ATR Sampling Accessory. IH NMR (400 MHz) and 13C NMR (100 MHz) spectra (all in CDCI3) were obtained in BRUKER DPX-400 spectrometer. ESIMS was recorded in a MICRO TM Q-TOF MICROMASS spectrometer. HPLC was carried out with WATERS HPLC instrument with a UV-VIS 486 detector set at 220 nm, using a Shim-pack PREP-ODS C-18 column (4.6 mm i.d. x 250 mm). Control of the equipment, data acquisition, processing, and management of chromatography information were performed by the WATERS 746 integrator. Chloroquine diphosphate was purchased from Sigma-Aldrich, USA. N,N-dimethylacetamide and PEG 300 were provided by Vetec. The solvents were from Quimex, Vetec, Carlo Erba and J. T. Baker.

Plant Materials The pieces of wood used in the present work were collected from the protected area inside of Zoo-Botanic Foundation of Belo Horizonte, Belo Horizonte, Minas Gerais state, Brazil. Voucher specimens (No. 22988) were deposited at the Instituto de Ci€mcias Biol6gicas Herbarium (BHCB), UFMG, Belo Horizonte, MG, Brazil.

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Extraction and Isolation The wood ofA. crassifZora was dried at 40°C, weighted (1166.00 g), grounded and extracted at room temperature with ethanol which was removed under vacuum to give the crude extracts (ACWF01; 150.9 g). Part of this extract (130.60 g) was dissolved in ethanoVwater (7:3) and successively extracted with hexane and chloroform. Mter solvent removal, the hexane (ACWF02), chloroform (ACWF03) and hydroalcoholic (ACWF04) fractions were obtained. The fraction ACWF03 was extracted with acid aqueous solution pH 4 followed by chloroform resulting in organic layer (ACWF03 0) and an acid aqueous layer (ACWF03 A). The acid aqueous phase was alkalinized and extract with chloroform, which after solvent removal yielded an alkaloidic fraction (ACW A). The ACWF03 0 was partitioned between hexane and MeOH-H 20 (9:1), furnishing the hydroalcoholic (ACWF05) and hexane (ACWF06) fractions. The ACWF05 0 fraction was submitted to biological guided fractionation on silica gel, followed by gel filtration on Sephadex LH-20 and preparative reverse phase HPLC with acetonitrilewater gradients, furnishing the acetogenins 1 (2.0 mg), 2 (2.0 mg), and 3 (1.5 mg). The alkaloidic fraction (ACW A) was fractionated on silica gel column affording the alkaloids 4 (3.0 mg), and 5 (2.5 mg). The ACWF02 fraction after separation by silica gel column chromatography eluted with hexane, dichlorometane, acetyl acetate and methanol gradients afforded the compounds 6 and 7 (7.0 mg) as a mixture.

Biological Evaluation Brine Shrimp Test Artemia salina encysted eggs (10 mg) were incubated in 100 mL of seawater under artificial lighting at 28°C, pH 7-8. After incubation for 24 h, nauplii were collected with a Pasteur pipette and kept for an additional 24 h under the same conditions to reach the metanauplii stage. The samples (triplicate) to be assayed were disolved in dimethylsulfoxide (DMSO) (2 mg/400 ~ or 2 mg/1000 ilL) and serially diluted (10, 20, 30 and 50 !Jll5mL) in seawater. About 10-20 nauplii were added to each set oftubes containing the samples. Controls containing 50 ilL of DMSO in seawater were included in each experiment. As a positive control, lapachol dissolved in DMSO was used. Twenty four h later, the number of survivors was counted, recorded and the lethal concentration 50% (LC 50 ) and 95% confidence intervals were calculated by Probit analysis (Finney, 1976). Antibacterial Assay The following bacterial strains were used: Bacillus subtilis ATCC 6633, Escherichia coli ATCC 25922, Micrococcus luteus ATCC 9341, Pseudomonas aeruginosa ATCC 27835 and Staphylococcus aureus ATCC 25923. They were cultured on Miller-Hinton agar at 37°C for 48 h. Antibacterial activity was assayed by agar diffusion method (Bauer et al., 1966). Dimethylformamide

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(DMF) was used for extracts solubilization. Sterile 6 mm diameter filter paper discs were impregnated with 1 mL of each extract (100 llg/mL). Inhibition zones surrounding the discs were measured after 24 and 48 h of bacterial growth. Chloramphenicol (30 J..lg/disc) was used as positive control and discs containing only DMF were used as negative control. Inhibition zone diameters around each disc (diameter ofinhibition zone minus diameter of the disc) were measured and recorded at the end of incubation time. The experiment was carried on in triplicate and an average zone was calculated from all measurements. Only inhibition zones equal or bigger than 7 mm (observed after 24 h) were considered of interest.

Antimalarial Activity in P. berghei-Infected Mice. Animal experiments were carried out according to the Principles of Laboratory Animal Care and legislation in force in Brazil and the protocol was approved by the animal ethical committee of the University. Female outbred Swiss albino mice weighing 20 to 24 g were supplied by the Animal Facility of Universidade Federal de Ouro Preto. They were kept in a normal diurnal cycle and had free access to food and water throughout the experiments. The classical 4-day suppressive test adapted from Peters et al. (1986) for determination of antimalarial activity against chloroquine sensitive Plasmodium berghei NK65 strain in Swiss albino mice was used for monitoring in vivo activity of the extracts and fractions. An infective inoculum was prepared from a previously infected donor mouse with rising parasitemia (20%). On day 0 the mice were infected intravenously with a million of infected erythrocytes of P. berghei in 0.2 mL of phosphate-buffered saline. They were randomly divided in groups of 5 mice and treated by the intraperitoneal route once daily (1.25; 12.5, 250 and 500 mg kg-I) with different extracts or fractions of A. crassiflora for four consecutive days (days 0 to 3). The extracts and fractions were dissolved in N,Ndimethylacetamide and polyethyleneglycol (PEG 300) at 1:2 proportion and further diluted 15-fold in saline to obtain the desired concentration in 0,3 mL for injection. Control groups received 0.2 mL of phosphate-buffered saline (untreated) or chloroquine diphosphate (60 mg/kg/day) as positive control. Thin blood smears were made from tail blood on days 4, 7, and 16 after infection. The parasitemia levels were assessed on Giemsa-stained thin blood smears. At least 3000 cells were checked to calculate parasitemia percent. Drug activity was determined on the basis of average parasitemia per group of mice. The percent reduction of parasitemia in treated groups as compared to untreated groups was calculated as follows: % parasitemia in control group (Pcg) - % parasitemia in test group/Pcg x 100.

Statistical Evaluation All RBC counts and parasitemia levels are expressed as mean values ± standard deviations. The parasitemia data were analysed by using the one-

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way analysis of variance (ANOVA) test using Prisma® 4.0 software. Mean survival times were compared by using Student's t test, considering a probability of 5% to be significant. RESULTS AND DISCUSSION

Isolation & Characterization The molecular formula of 1 was determined as C37H6607 from the [M+H]+ peak at mlz 623.53 in the electro spray ionization mass spectrum (ESIMS). A positive Kedde test (Alali et al., 1999) and IR absorptions at 3400, 2920, 2850,1745,1650 cm-l suggested 1 to be an annonaceous acetogenin bearing an a,~-unsaturated y-Iactone moiety. The presence of a typical a, ~­ unsaturated-y-Iactone, without an hydroxyl group at C-4 (Alali et al., 1999; Kim et al., 2001) was suggested by the signals at 0 (H) 6.98 (1 H, td, J H-35/ H-36 = J H-35/H-3 = 1.40 Hz, H-35); 4.99 (1 H, qdd, J H-36/H-37 = 6,80 Hz; J H-35/ H-36 = J H-36/H-3 = 1.40 Hz, H-36); 1.41 (1 H, d, J H-36/H-37 = 6.80 Hz, H-37) e 2.26 (2 H, tt, J H-3/H-4 = 7.6 Hz; J H-3/H-35 = J H-3/H-36 = 1.4 Hz, H-3) in the lH_ NMRspectrum (Table 1), which showed correlations with carbons in the HMQC and HMBC spectra, permitting the assignment of carbons signals o (C) 173.94 (C-1), 148.91 (C-35), 134.40 (C-2), 77.42 (C-36) and at 19.20 (C-37). The presence of an adjacent bis-THF rings system with two flanking OH groups was suggested by the four carbon signals between 0 83.30 and 82.14, which were correlated to the proton signals at 0 3.91 (5H) in the HMQC spectrum. The one-bond lH_13C correlations detected in the HMQC spectrum, along with those observed in COSY, permitted the assignment ofthe signals at 0 74.00 and 71.80 to oxygenated methine carbons and at 0 3.42 and 03.91 to the hydrogen adjacent to the THF-rings. COSY spectrum showed correlation between these signals at 0 3.91 (5H) and the multiplet at 0 3.42 (lH). The presence of a third hydroxyl group in the hydrocarbon chain was suggested by the signals at 0 3.60 (1H) and 0 71.40 (in lH NMR and l3C NMR spectra, respectively). The placement of THF rings and the additional hydroxyl group (0 3.60 and 071.40) was determined by a close examination of the ESIMS fragmentation of 1 after induced collision of the ion at mlz 623.53. The hydroxyl group was located at C9 on basis of fragments at mlz 439, 411 e 181 in ESIMS of 1 (Figs 1,2). Only the relative stereochemistry at C-11/C-12, C-15/C-16 and C-19/C-20 was determined by comparing the lH_ and l3C-NMR data to those of model compounds of known relative stereochemistry (Cave et al., 1997; Jossang et al., 1990; Sahai et al., 1994). This comparison showed the relative configurations between the carbons C-11 to C-20 being as threo-trans-threo-trans-erythro considering the similarity ofthe lH_ and l3C-NMR data of 1 with those of the bullatacin-type of acetogenins (Zeng et al., 1996; Sahai et al., 1994). The planar structure of this adjacent bis-THF acetogenin annofolin (1) with a hydroxyl group in C-9, to the best of our knowledge, was described by our

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Fig Contd.

22

~

23

6

7

Fig 1. Structures of the compounds isolated from Annona crassiflora wood

The planar structure of the ACM5 (2) is similar to annofolin (1) differing only in the relative stereochemistry around the THF rings that was determined as threo-trans-threo-trans-threo by IH NMR data (Table 1). Table 1. 1Ha and 13C NMR spectral data of anofolin (1), and 1Ha NMR data of ACM5 (2) and ACM6 (3) Annofolin

Carbon IH(J)

1 2 3 4 5-7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

25-33 34 35 36 37

2.26 tt (7.60, 1.40) 1.54m 1.26-1.30 m 1.40m 3.60m 1.40m 3.42m 3.91m 1.54-2.00 1.54-2.00 3.91m 3.91m 1.54-2.00 1.54-2.00 3.91m 3.91m 1.40m 1.26m 1.26-1.30 m 1.26-1.30 m 1.26-1.30m 0.89 t (6.60) 6.98 d (1.40) 4.99 qd (6.80, 1.40) 1.41 d (6.80)

a CDCl3, 400 MHz. (J in Hz)

13C 173.9 134.4 25.0 27.0 22.5-29.5 32.0 71.4 32.0 74.0 83.2 27.0 28.8 82.0 82.0 28.8 28.3 82.7 71.8 32.0 22.5-29.5 22.5-29.5 22.5-29.5 22.5-29.5 14.0 148.9 77.4 19.2

ACM5

ACM6

IH(J)

IH(J)

2.26 tt (7.66, 1.52) 1.58m 1.26m 1.40m 3.60m 1.40m 3.41m 3.86-3.91 m 1.54-2.00 1.54-2.00 3.86-3.91m 3.86-3.91m 1.54-2.00 1.54-2.00 3.86-3.91 m 3.41m 1.40 1.26m 1.26m 1.26m 1.26m 0.89 t (6.40) 6.98 d (1.52) 4.99 qd (6.80, 1.52) 1.41 d (6.80)

2.26 tt (7.66,1.00) 1.56m 1.26m 1.26m 1.26m 1.26m 1.26m 1.39m 3.61m 1.39m 3.47m 3.93m 1.56-1.97 m 1.56-1.97 m 3.93m 3.93m 1.56-1.97m 1.56-1.97 m .93m 3.93m .26-1.39 m 0.88 t (6.80) 6.99 d (1.00) 4.99 qd (6.40, 1.00) 1.41 d (6.40)

Annona crassiflora Wood Constituents

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+

37

3

o _____ ..J

(227)

~

-H20

209

Fig 2.

(367) +-H20

349

-----, -3H 0

(441)~ 387

~ -H2

439

Diagnostic ESIMS fragment ions (m/z) for annofolin (1). Peaks in parentheses were not observed

The planar structure of ACM6 (3) was suggested based on the IH_ NMR (Table 1), COSY and ESI mass spectra. The signals at 86.99 (lH, d, H35); 4.99 (lH, qd, H-36); 1.41 (3H, d, H-37) and 2.26 (2H, tt, H-3), indicated the presence of a a, ~-unsaturated-y-Iactone , without an hydroxyl group at C-4 (Alali et al., 1999). The signals at 8 3.47 (lH, m, H-15); 3.61 (lH, m, H-13) e 3.93 (5H, m, H-16, 19,20,23 e 24) are characteristics of de hydrogens of oxygenated carbons, with the signal at 8 3.61 assigned to a hydroxyl group in the hydrocarbon chain (Hisham et al., 1991) and the others assigned to a bis-THF system with two flanking hydroxyl groups. The relative stereochemistry from C-15 to C-24 was suggested as threo-trans-threo-transerythro considering the similarity of the 1H - and 13C-NMR data of 3 with 1 and those of the bullatacin-type of acetogenins (Zeng et al., 1996). The ESIMS data revealed the presence of one acetogenin with molecular formulae of C37H6607. Based on analysis of fragment ions obtained after induced collision ofthe ion mlz 623.40 [M + Hj+ the the system bis-THF a, a'-dihydroxylated was located between C-15 and C24, and the third hydroxyl group at C-13 (Fig 3). The structures of the alkaloids aterospermidine (4) and liriodenine (5) and the steroids ~-sitosterol (6) and stigmasterol (7), were confirmed by one and bidimensional IH_ and 13C-NMR data and MS measurements, compared to those of the literature (Guinadeau et al., 1975; Hossain et al., 1995; Castro et al., 1996; Goulart et al., 1993). This is the first report of the alkaloid presence in the wood of Annona crassiflora, and the first report of aterospermidine in this species.

302

Compo Bio. Nat. Prod., Vol. 2 - Efficacy, Safety & Clinical Evaluation I 363

293

t-

t-

H2O

(381)

265

t

H2O

(311)

-H2

(267)

237

,r----,

23

OH

'D y5 '

"""'20! 19

0"

37

: ,, ,

0\\\

\".,

yl3 !.(CH 2 :

16

:

, ,,

I

OH

I

OH:

o

(311)

+-H,O 293

Fig 3. Diagnostic ESIMS fragment ions (m/z) for annofolin (3). Peaks in parentheses were not observed

Biological assays The crude extract (ACWF01) and the fractions ACWF02, ACWF03, ACWF03 0, ACWA, ACWF05, ACWF06 showed good bioactivity in brine shrimp lethality test with the DL50 range from 1.29 to 30 J..I.g/mL (Table 3), This is in accordance with the presence of annonaceous acetogenis as well as aporfine alkaloids.

Plasmodium berghei NK-65 strain, sensitive to chloroquine (Peters et al., 1986), was used for antimalarial evaluation in vivo. This strain is known to induce high mortality in mice, providing a good model to estimate survival and antimalarial efficacy in reducing parasitemia, and is sensitive to all currently used antimalarial drugs. It was kindly supplied frozen in nitrogen by Prof. E.M. Braga, Malaria Laboratory, Parasitology Department, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil. The antimalarial activity was determined using the four-day suppressive test in P. berghei infected mice. Doses of the extract and fractions of 500 mg/kg, 250 mg/kg, 12.5 mg/kg and 1.25 mg/kg were tested. The crude extract caused 100% of death in a dose of 500 mg/kg, and 60 % of death with 250 mg/kg in mice, by intraperitoneal route in the first 24 h after administration. Table 2 summarizes the effects of extracts and fractions of A. crassiflora at concentrations of 1.25 and 12.5 mg/kg/day in vivo. The results showed that alkaloidic fraction (ACWA) has a significant antimalarial activity compared with untreated control (p<0.05) and a mean survival time

Annona crassiflora Wood Constituents

303

greater than the control indicating the smaller toxicity of this fraction. However, the other fractions especially hexane fraction (ACWF02) exhibits higher toxicity even at the lower doses used in this protocol. In general, the percentage of parasitemia reduction increases with the dose and particularly with the time, suggesting that the pharmacokinetic profile ofthe substances responsible for the activity possesses long half-lives in mice. No fraction or extract was able to suppress 100% parasitemia (Table 2). However all the extracts and fractions produced some activity reducing the parasitaemia in 13 to 79% range. As expected the P. berghei NK65 strain was chloroquinesensitive parasite and 60 mg/kg/day had a suppressive activity of 100%. The solvents DMAIPEG/saline had no significant effect (p>0.05) on parasitemia and on the mean survival time (data not shown) compared to controls. In the antibacterial activity test by disc diffusion, the alkaloidic fraction, ACW A, showed good results against pathogenic bacteria Staphylococcus aureus, Streptococcus pyogenes, Salmonella typhimurium and Escherichia coli (Table 3). This is the first report of phytochemical contituents of Annona crassiflora wood with evaluation in different biological activity tests. The results indicated this plant as a potential source of lead compounds for the development of antitumoral, pesticide, antimicrobial and antimalarial drugs. Table 2. Mean Parasitemia (%), mean survival time and parasitemia reduction (%) in P. berghei NK65 infected mice treated with extracts and fractions of Annona

crassiflora Codes

Fractions

Dose mgIKgI day

MST# Days ±SD

Mean Parasitemia(%)# D4

Parasitemia Reduction

D7

(%)

(days 417) Control Not treated Chloroquine

16.2 ± 3.3 >30 14.3 ± 4.0§

ACWF01

60.0 Crude extract 1.25

ACWF01 ACWF02

Crude extract 12.5 Hexanic 1.25

ACWF02

Hexanic

ACWF05

Hydroalcoholic 1.25 Hydroalcoholic 12.5

13.3 ± 4.8§

Alkaloidic

1.25

20.8 ± 4.8§

Fraction from ACWF030

1.25

14.3 ±

ACWF05 ACWA F1-G24 F1-G24

12.5

Fraction from 12.5 ACWF030

14.4 ± 3.5 0 12.6 ± 1.7§

24.6 ± 3.3 0 15.9 ± 0.7*§ 8.9 ± 1.7*§ 11.4 ± 0.7*§

14.8 ± 4.4 9 13.3 ± 2.1§ 11.6 ± 2.0§ 10.2 ± 7.8§ 11.1 ± 2.6§ 16.8 ± 4.1§ 4.0~

11.5 ± 4.7§

10.4 ± 0.5*~ 5.0 ± 0.2a

10.6 ± 2.2*§ 13.5 ± 0.6*§ 12.0 ± 1.8§ 12.4 ± 0.6*§ 7.2 ± 1.4*§

100/100 13/35 38/54 19/58 23/79 26/45 16/49

7.0 ± 1.3*§ 9.2 ± 2.1H 16.8 ± 1.3*§

50/71 36/32

8.5 ± 2.1 *§

41/67

8.2 ± 2.7*§

#n=5; MST: mean survival time; *different from control (p< 0.05); chloroquine group (p< 0.05) using student t test, "two mice.

§

different from

304

Camp. Bio. Nat. Prod., Vol. 2 - Efficacy, Safety & Clinical Evaluation I

Table 3. Brine shrimp lethality and antimicrobial data for the extract and selected fractions from A. crassiflora

Compound

BSTb (llglmL)

ACWF01 27.7 ACWF02 3.8 ACWF03 2.9 ACWF05 4.6 ACWF06 1.6 ACWF030 1.8 ACW A Chloramphenicol a

b

Diametera (mm) of inhihition zone of bacterial growth

S. aureus

S. typhimurium S. pyogenes

E. coli

24h

48h

24h

48h

24h 48h

24h 48 h

NDc 7.0 ND ND 8.0 ND 12.0 20.0

ND 6.0 ND ND 7.0 ND 12.0 22.0

ND 9.0 11.0 10.0 11.0 8.0 14.0 22.0

ND 8.0 10.0 10.0 11.0 8.0 14.0 22.0

ND ND 11.0 11.0 15.0 9.0 14.0 20.0

ND 9.0 ND ND ND ND 10.0 ND

ND ND 11.0 11.0 14.0 9.0 13.0 19.0

ND 9.0 ND ND ND ND 10.0 ND

Diameter of inhibition zone minus diameter of the disc after 24 and 48 h observed for the tested extracts, fractions (100 Ilg/mL) and chloramphenicol (30 Ilg/disc). Brine Shrimp Lethality test. C Not detected.

ACKNOWLEDGEMENTS The authors thank F APEMIG for financial support (grant CDS-853/06), CNPq and PRPq for a scholarship to M.A.G. We also thank Dr. Jacqueline Aparecida Takahashi (Chemistry Department/UFMG) for arranging for the antimicrobial assays and Dr. Andreia M. Amaral (ICBIUFMG) for providing the bacteria strains. The technical support of Elaine A. Leite is also acknowledged. We also grateful to Albina Carvalho de Oliveira Nogueira for collecting the plant material.

REFERENCES Alali, F.Q., Liu, X.-X. and McLaughlin, J.L. (1999). Annonaceous acetogenins: recent progress. J. Nat. Prod., 62: 504-540. Bauer, A.W., Kirby, W.M.M., Sherriss, J.C. and Turck, M. (1966). Antibiotic susceptibility testing by a standardized single disk method. American Journal of Clinical Pathology, 45: 493-496. Bermejo, A., Figadere, B., Zafra-Polo, M-C., Barrachina, I., Estornell, E. and Cortes, D. (2005). Acetogenins from Annonaceae: recent progress in isolation, sythesis and mechanisms of action. Nat. Prod. Rep., 22: 269-303. Cave A., Cortes D., Figadere B., Laurens A. and Pettit G.R. (1997). In: "Progress in the Chemistry of Organic Natural Products", Springer-VerJag/Wien, Austria, pp. 81-288. Castro, I., Villegas, J.R., Soeder, B. and Castro, O. (1996). Oxoaporphine alkaloids from Annona purpurea wood. Fitoterapia, 67: 181-182. Fang, X-P., Rieser, M.J., Gu, Z-N., Zhao, G.-X. and McLaughlin, J.L. (1993). Annonaceous acetogenins: An updated review. Phytochem. Anal., 4: 49-67. Fisher, D.C.H., Gualda, N.C.A., Bachiega, D., Carvalho, C.S., Lupo, F.N., Bonotto, S.V., Alves, M.O. Yogi, A., Santi, S.M.D., Avila, P.E., Kirchgatter, K. and Moreno, P.R.H. (2004). In vitro screening for anti plasmodial activity of isoquinoline alkaloids from Brazilian plant species. Acta Tropica, 82: 261-266. Garavito, G., Rincon, J., Arteaga, L., Hata, Y., Bourdy, G., Gimenez A., Pinzon, R. and Deharo, E. (2006). Antimalarial activity of some Colombian medicinal plants. J. Ethnopharm., 107: 460-462.

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Goulart, M.O.I., Sant'ana, A.E.G., Lima, R.A. and Cavalcante, S.H. (1993). Fitoconstituintes quimicos isolados de Jatropha ellhpt~ca. Atribui~ao dos deslocamentos quimicos dos atomos de carbono e hidrogenio dos triterpenos de jatrofolonas AeB. Quimica Nova, 16(2): 95-100. Guinaudeau, H. Leboeuf, M. and Cave, A. (1975). Aporphine alkaloids. (Lloydia) J. Nat. Prod., 38: 275. Hisham A., Pieters, L.A.C., Claeys, E.E., Dommisse, R. and Vlietinck, A.J. (1991). Squamocin-28-one and panalicin, two acetogenins form Uvaria narum. Phytochemistry, 30: 545-548. Hocquemiller, R., Cave A., Jacquemin, H., Touche, A. and Forgacs, P. (1982). Alcaloides des Annonacees, 36: alcaloides de l'Annona crassiflora Mart. Plantes Medicinales et Phytothtirapie, 16: 4-6, Hossain, M.S., Ferdous, A.J. and Hasan, C.M. (1995). Alkaloids from the stem bark of Desmos longiflorus. F~toterapia, 66: 463-464. Jossang, A., Dubaele, B. and Cave, A. (1990). Deux nouvelles acetogenins monotetrehydrofuranniques cytotoxiques: l'annomonicine et la montanacine. Tetrahedron Lett., 31: 1861-1864. Lima, L.A.R. dos S. (2007). Estudo Quimico e Avalia~ao Bio16gica das Sementes de Annona cornifolia A. St. -Hil. (Annonaceae) PhD Thesis, UFMG, Belo Horizonte, MG, pp. 317. Lorenzi, H. and Matos, F.J.A. (2002). Plantas medicinais no Brasil: nativas e ex6ticas cultivadas. Nova Odessa, SP: Instituto Plantarum. Peters, W., -Lin, L. Ze, Robinson, B.L. and Warhurst, D.C. (1986). The chemotherapy of rodent malaria, XL. Ann. Trop. Med. Parasitol., 80: 483-489. Pimenta, L.P.S. (1995). Estudo Quimico Bio-monitorado das sementes de Annona crassiflora objetivando 0 isolamento de acetogeninas tetra-hidrofuranicas. PhD Thesis, UFMG, Belo Horizonte, MG, pp. 268. Pimenta, L.P.S., Boaventura, Maria Amelia Diamantino, Sun, N.-J., Cassady, J.M. and Oliveira, A.B. (1996). Araticulin, a Bis-tetrahydrofuran polyketide from Annona crass~flora seeds. Phytochemistry, 42: 705-707. Pimenta, L.P.S., Prates, H.T., Viana, P.A. and Boaventura, M.A.D. (2000), Insecticide action of ethanolic extract from Annona crassiflora seeds against Spodoptera frugiperda In: XXI International Congress of Entomology, 2000, Foz do Iguassu - Parana. Abstract Book I, 1: 1035-1035. Pimenta, L.P.S., Nascimento, F.C., Assun~ao, A.C. and Boaventura, M.A.D. (2001). Laurifolin, a novel acetogenin from Rollinia laurifoha leaves. Tetrahedron Lett., 42: 8433-8434. Pimenta, L.P.S., Pinto, G.B., Takahasi, J.A., Silva, L.G.F. and Boaventura, M.A.D. (2003). Biological screenig of annonaceous Brazilian medicinal plants using Artemia salina (brine shrimp test). Phytomedicme, 10: 209-212. Pimenta, L.P.S., Lima, L.A.R. dos S. and Boaventura, M.A.D. (2008). Submitted for publication. Rupprecht, J.K., Hui, Y.H. and Mclaughlin, J.L. (1990). Annonaceous acetogenins: a review. J. Nat. Prod., 53: 237-278. Sahai, M., Singh, S., Sing, M., Gupta, Y.K., Akashi, S., Yuji, R., Hirayama, K., Asaki, H., Araya, H., Hara, N., Eguchi, T., Kakinuma, K. and Fuji Oto, Y. (1994). Annonaceous acetogenins from the seeds of Annona squamosa. Adjacent bis-tetrahydrofuranic acetogenins. Chem. Pharm. Bull., 42: 1163-1174. Santos, L.P., Boaventura, M.A.D. and Oliveira, A.B. (1994). Crassiflorina, uma acetogenina tetra-hidrofuranica citot6xica de annona crassiflora (araticum). Quimica Nova , 17: 387-391. Santos, L.A.R. dos, Pi menta, L.P.S., Boaventura, M.A.D. (2006). Cornifolin, a new bistetrahydrofuran annonaceous acetogenin from Annona cornifolia. Bwchemical Systematics and Ecology, 34: 78-82. Takahashi, J.A., Pereira, C.R., Pimenta, L.P.S., Boaventura, M.A.D. and Silva, L.G.F.E. (2006). Antibacterial activity of eight Brazilian Annonaceae plants. Nat. Prod. Rep., 20: 21-26. Zeng, L., Ye, Q., Oberlies, N.H., Shi, G., Gu, Z.-M., He K. and McLaughlin, J.L. (1996). Recent Advances in Annonaceous Acetogenins. Nat. Prod. Rep., 275-293.

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16 Pharmacokinetics of Active Constituents of Rhodiola rosea SHR-5 Extract A. PANOSSIAN2,*, A. HOVHANNISYAN 1, H. ABRAHAMYANl,

E. GABRIELYAN 1 AND G. WIKMAN 2

ABSTRACT The active constituents of Rhodola rosea extract SHR-S of the popular plant adaptogen Rhodiola rosea are tyrosol, rhodioloside and rosavi